Substituted benzodiazoliums as ENaC inhibitors

ABSTRACT

Compounds of general formula (I) wherein R1, R2, R3, R4, R5 and X are as defined herein are inhibitors of the epithelial sodium channel (ENaC) and are useful for the treatment or prevention respiratory diseases and conditions, skin conditions and ocular conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/462,794, filed on May 21, 2019, incorporated by reference in itsentirety, which is a U.S. National Stage filing under 35 U.S.C. § 371 ofInternational Application No. PCT/GB2017/053499, filed on Nov. 22, 2017,which claims priority to United Kingdom Application No. GB 1619694.1,filed on Nov. 22, 2016.

TECHNICAL FIELD

The present invention relates to novel compounds which have activity asinhibitors of the epithelial sodium channel (ENaC). The invention alsorelates to the use of these compounds in treating diseases andconditions modulated by ENaC, particularly respiratory diseases andconditions, methods of preparing the compounds and pharmaceuticalcompositions containing them.

BACKGROUND

Humans can inhale up to 12,000 L of air each day and with it comes thepotential for airborne pathogens (bacteria, viruses, fungal spores). Toprotect against these airborne pathogens, the lung has evolved innatedefence mechanisms to minimise the potential for infection andcolonisation of the airways. One such mechanism is the mucus clearancesystem, whereby secreted mucus is propelled up and out of the airways bythe coordinated beating of cilia together with cough clearance. Thisongoing ‘cleansing’ of the lung constantly removes inhaled particles andmicrobes thereby reducing the risk of infection.

In recent years it has become clear that the hydration of the mucus gelis critical to enable mucus clearance (Boucher 2007; Matsui et al,1998). In a normal, healthy airway, the mucus gel is typically 97% waterand 3% solids under which conditions the mucus is cleared by mucociliaryaction. The hydration of the airway mucosa is regulated by thecoordinated activity of a number of ion channels and transporters. Thebalance of anion (Cl⁻/HCO₃ ⁻) secretion mediated via the Cystic FibrosisTransmembrane Conductance Regulator (CFTR) and the Calcium ActivatedChloride Conductance (CaCC; TMEM16A, also known as Ano1) and Na⁺absorption through the epithelial Na⁺ channel (ENaC) determine thehydration status of the airway mucosa. As ions are transported acrossthe epithelium, water is osmotically obliged to follow and thus fluid iseither secreted or absorbed. As ions are transported across theepithelium, water is osmotically obliged to follow and thus fluid iseither secreted or absorbed.

In respiratory diseases such as chronic bronchitis and cystic fibrosis,the % solids of the mucus gel is increased as the hydration is reducedand mucus clearance is reduced (Boucher, 2007). In cystic fibrosis,where loss of function mutations in CFTR attenuates ability of theairway to secrete fluid, the % solids can be increased to 15% which isbelieved to contribute towards the plugging of small airways and failureof mucus clearance. Furthermore, in cystic fibrosis an increase in ENaCactivity has been reported by several groups (Knowles et al, 1983;Middleton et al, 1993) and this increase in ENaC function has been shownto correlate with disease severity (Fajac et al, 2004; Leal et al,2008). Strategies to increase the hydration of the airway mucus includeeither the stimulation of anion and thereby fluid secretion or theinhibition of Na⁺ absorption. To this end, blocking the activity of ENaCwill inhibit Na⁺ absorption and therefore increase fluid accumulation inthe airway mucosa, hydrate mucus and enhance mucus clearance mechanisms.

ENaC is expressed in renal, colonic, corneal, sweat duct and respiratoryepithelia where it forms a low conductance channel (˜4 pS) with aselectivity for Na⁺ over K⁺ of approximately 10-fold (Kellenberger2002). Loss and gain of function mutations in the channel can causehuman disease including pseudohypoaldosteronism type 1 (PHA1), a saltwasting disease (Chang et al, 1996), and Liddles's syndrome, a diseaseassociated with salt retention and hypertension (Botero-Velez et al,1994). Of particular note to lung physiology is the observation thatpatients with PHA1 loss-of-function mutations in ENaC have an enhancedrate of airway mucociliary clearance (MCC) compared with the normalhealthy population, typically 3-4 fold faster (Kerem et al, 1999).Furthermore, the upper airways of these patients appear to be ‘wet’ andhave extra-hydration compared to normal. These observations furthersupport the salient role that ENaC plays in the human airway in theregulation of hydration and the therapeutic benefit that blocking ENaCin the airway could deliver in terms of enhancing MCC and innatedefence.

Amiloride, a small compound blocker of ENaC, has been demonstrated toincrease MCC in both healthy controls and also patients with CF, furthersupporting the physiological significance of this mechanism (App et al,1990). However, the lack of a robust effect of inhaled amiloride onclinical endpoints (Bowler et al, 1995; Graham et al, 1993; Knowles etal, 1990; Pons et al, 2000) was ascribed to the short duration of actionof this compound in the lungs (Noone et al., 1997). Novel ENaC blockers,specifically designed for a long duration of action in the airway aretherefore predicted to acutely provide an extended enhancement of MCCwith resulting clinical benefit in the longer term.

A challenge with the design of inhaled ENaC blockers for the treatmentof respiratory diseases has been the potential for the renal-based sideeffect of hyperkalaemia (Perazela et al., 2000). ENaC is expressed inthe cortical collecting duct of the kidney epithelium and block of thechannel here can lead to a systemic accumulation of K⁺. For this reason,it is desirable that an inhaled ENaC blocker avoids renal exposurefollowing absorption from the lung. This could be achieved througheither a high lung retention of ENaC blocker therefore enabling only alow dose to be administered or through the design of a compound thatwill be rapidly transformed to an inactive metabolite before it reachesthe kidney.

ENaC blockers have also been implicated in the hydration of skin and thesurface of the eye (Frateschi et al, 2010; Thelin et al, 2012).

Several ENaC blockers are known. For example, WO 2011/113894 relates tocompounds which are said to be of use for treating inflammatory orobstructive diseases of the airways or for promoting mucosal hydration.The compounds are of the formula:

where A is N or CR^(4a) and R² is haloalkyl. None of the compoundsexemplified in this document contain a benzimidazole moiety.

WO 2011/079087 relates to compounds of the formula:

WO 2015/007516, WO 2015/007517 and WO 2015/007519 all relate tocompounds of the formula:

WO 2016/113168, WO 2016/113167 and WO 2016/113169 relate to compounds ofthe formula:

WO 2016/113170 relates to compounds of the formula:

The compounds described in these documents all contain a6-halo-3,5-diaminopyrazine group and this group is also a structuralfeature of the ENaC inhibitors disclosed in numerous other documentsincluding WO2013/0664450, WO2013/092674, WO2014/044849, WO 2014/177469,WO 2015/003958, WO2015/018754, WO 2011/028740, WO 2007/071396, WO2007/071400, WO 2008/135557, WO 2009/074575, WO 2009/138378, WO2009/150137 and WO 2012/035158 Other documents relating to pyrazinederivatives with ENaC inhibitor activity include WO 2015/003083, WO2004/073629, WO 03/070184, WO 03/070182, WO 2006/022935, WO 2007/018640,WO 2008/124491, WO 2009/139948, WO 2005/044180, WO 2005/016879, WO2005/018644, WO 2005/025496, WO 2005/034847 and WO 2013/181232. However,every compound exemplified in these documents contains a6-halo-3,5-diaminopyrazine group and it is therefore clear that apyrazine ring with amino substituents at the 3- and 5-positions and6-halo substituent was, until recently, considered essential for ENaCblocking activity. Some more recent documents relate to ENaC blockingcompounds in which the 5-amino group is not present. For example, WO2017/028926 relates to ENaC inhibiting compounds of the formula:

and WO 2017/028927 relates to ENaC inhibiting compounds of the formula:

Our earlier application PCT/GB2017/051815 also relates to compoundscomprising a pyrazine group with a single amino substituent at the3-position, with most of these compounds also having a substituent atthe pyrazine 6-position.

SUMMARY

The present inventors have surprisingly discovered that compounds withalternative structures to the 6-halo-3,5-diamino pyrazine or6-substituted-3-aminopyrazine also have ENaC blocking activity and mayhave beneficial properties compared with the known compounds,particularly in relation to the ADME (Absorption, Excretion,Distribution and Metabolism) properties.

In the present invention there is provided a compound of general formula(I) including all tautomeric forms, all enantiomers and isotopicvariants and salts thereof:

-   -   wherein    -   X⁻ is an anion;    -   R¹ is:    -   i. H or halo; or    -   ii. -L¹R¹⁰, wherein    -   L¹ is:    -   —Z¹—, -Q¹-, —Z¹Q¹-, -Q¹Z¹—, —Z¹Q¹Z²—, -Q¹Q²-, -Q¹Q²Z¹—,        -Q¹Q²Z¹Q³Z²—, —Z¹Q¹OQ²OQ³-; —OZ¹—, —OQ¹-, —OZ¹Q¹-, —OQ¹Z¹—,        —OZ¹Q¹Z²—, —OQ¹Q²-, —OQ¹Q²Z¹—, —OQ¹Q²Z¹Q³Z²—, —OZ¹Q¹OQ²OQ³-;    -   —Z¹N(R⁷)Z²—, -Q¹Z¹N(R⁷)Z²—, —Z¹N(R⁷)Z²Q¹-, -Q¹Z¹N(R⁷)Z²Q²Z³—;    -   —Z¹O(CH₂CH₂O)_(n)Z²—, —Z¹O(CH₂CH₂O)_(n)Q¹-,        —Z¹O(CH₂CH₂O)_(n)Z²Q¹, —Z¹O(CH₂CH₂O)_(n)Q¹Z²—,        -Q¹Z¹O(CH₂CH₂O)_(n)Z²—, -Q¹Z¹O(CH₂CH₂O)_(n)Q¹-,        -Q¹Z¹O(CH₂CH₂O)_(n)Z²Q¹, —Z¹O(CH₂CH₂O)_(n)Z²Q¹Z³—;    -   —C(O)Z¹—, —C(O)Q¹-, —C(O)Z¹Q¹-, —C(O)Z¹Q¹Z²—, —C(O)Q¹Z¹—,        —C(O)Q¹Q²-, —C(O)Q¹Q²Z¹—, —C(O)Q¹N(R⁷)C(O)Z¹—,        —C(O)Q¹N(R⁷)C(O)Z¹Q²-, —C(O)Q¹N(R⁷)C(O)Z¹Q²Q³-,        —C(O)Q¹N(R⁷)C(O)Z¹Q²Z²—, —C(O)Z¹Q¹OQ²OQ³-;    -   —C(O)N(R⁷)Z¹—, —C(O)N(R⁷)Q¹-, —C(O)N(R⁷)Z¹Q¹-,        —C(O)N(R⁷)Z¹Q¹Z²—, —C(O)N(R⁷)Q¹Z¹—, —C(O)N(R⁷)Q¹Q²-,        —C(O)N(R⁷)Q¹Q²Z¹—, —C(O)N(R⁷)Z¹Q¹Q²Z²—,        —C(O)N(R⁷)Z¹O(CH₂CH₂O)_(n)Z²—, —C(O)N(R⁷)Z¹O(CH₂O)_(n)Z²—,        —C(O)N(R⁷)Z¹Q¹Z²N(R⁸)Z³—, —C(O)N(R⁷)Z¹N(R⁸)Z²—,        —C(O)N(R⁷)Q¹Z¹N(R⁸)Z²—, —C(O)N(R⁷)Z¹Q¹OQ²OQ³-,        —C(O)N(R⁷)Z¹Q¹OQ²OQ³Z²—;    -   —Z¹C(O)N(R⁷)Z²—, —Z¹C(O)N(R⁷)Q¹-, —Z¹C(O)N(R⁷)Z²Q¹-,        —Z¹C(O)N(R⁷)Q¹Z²—, —Z¹C(O)N(R⁷)Q¹Q²-, —Z¹C(O)Q¹-, —Z¹C(O)Q¹Z²—,        —Z¹C(O)Q¹Q²-, —Z¹C(O)N(R⁷)Q¹Q²Z²—, —C(O)OZ¹—, —C(O)OQ¹-,        —C(O)OZ¹Q¹-, —C(O)OZ¹Q¹Z²—, —C(O)OQ¹Z¹—, —C(O)OQ¹Q²-,        —C(O)OQ¹Q²Z¹—;    -   Q¹C(O)Q²-, Q¹C(O)Z¹—, -Q¹C(O)Q²Z¹—, Q¹C(O)Q²Q³-, Q¹C(O)Z¹Q²-,        Q¹C(O)Q²Q³Z¹—;    -   —C(═NR⁹)N(R⁷)Z¹—, —C(═NR⁹)N(R⁷)Q¹-, —C(═NR⁹)N(R⁷)Z¹Q¹-,        —C(═NR⁹)N(R⁷)Z¹Q¹Z²—, —C(═NR⁹)N(R⁷)Q¹Z¹—, —C(═NR⁹)N(R⁷)Q¹Q²- or        C(═NR⁹)N(R⁷)Q¹Q²Z¹—; wherein        -   each of Z¹, Z² and Z³ is independently C₁₋₁₂ alkylene, C₂₋₁₂            alkenylene, C₂₋₁₂ alkynylene any of which is optionally            substituted by one or more substituents selected from halo,            OH, C(O)NR¹⁵R¹⁶, C(O)OR¹⁵ and NR¹⁵R¹⁶;            -   each R¹⁵ and R¹⁶ is independently H or C₁₋₆ alkyl or R¹⁵                and R¹⁶ together with the nitrogen atom to which they                are attached may form a 5- or 6-membered heterocyclic                ring optionally containing one or more further                heteroatoms selected from N, O and S;        -   each of Q¹, Q² and Q³ is independently carbocyclyl,            heterocyclyl, aryl or heteroaryl any of which is optionally            substituted with one or more substituents selected from            halo, OH, C₁₋₄ alkyl, C₁₋₄ haloalkyl, C(O)NR¹⁵R¹⁶, C(O)OR¹⁵            and NR¹⁵R¹⁶, and, for cycloalkyl and heterocyclyl groups,            oxo, wherein R¹⁵ and R¹⁶ are as defined above; n is 1 to 6;        -   each R⁷ and R⁸ is independently selected from H and C₁₋₁₂            alkyl optionally substituted with one or more halo or OH            groups, or        -   when an R⁷ and an R⁸ or two R⁸ groups are attached to a            nitrogen atom they may, together with the nitrogen atom            combine to form a 5- or 6-membered heterocyclic ring            optionally comprising one or more further heteroatoms            selected from N, O and S;        -   R⁹ is H or C₁₋₆ alkyl;    -   R¹⁰ is H, —N(R⁷)R⁸, —N(R⁷)C(═NR⁹)N(R⁸)₂, —N(R⁷)—C(O)OR⁸, OR⁷ or        —C(O)OR⁷; or    -   a cationic group selected from —N(R⁷)—C(O)—(C₁₋₃        alkylene)-N⁺(R⁸)₃ and —N⁺(R⁸)₃, in which case, an additional        anion X⁻ will be required; and        -   R⁷, R⁸ and R⁹ are as defined above; or    -   iii. —R¹², —OR¹²—SO₂R¹², —C(O)OR¹², —C(O)NR¹²R¹³,        —C(═NR⁹)NR¹²R¹³, -Q¹R¹²—, -Q¹OR¹² -Q¹SO₂R¹², -Q¹C(O)OR¹²,        -Q¹C(O)NR¹²R¹³, -Q¹C(═NR⁷)NR¹²R¹³, -Q¹Q²R¹², -Q¹SO₂R¹²,        -Q¹Q²C(O)OR¹², -Q¹Q²C(O)NR¹²R¹³ or -Q¹Q²C(═NR⁹)NR¹²R¹³; wherein        -   Q¹ and Q² are defined as above; and        -   each R¹² and R¹³ is independently H, C₁₋₆ alkyl, C₂₋₆            alkenyl, C₂₋₆ alkynyl, C₃₋₈ cycloalkyl or C₃₋₈ heterocyclyl,            any of which is optionally substituted by one or more            substituents selected from halo, OR⁷, C(O)OR⁷, —N(R⁷)R⁸ and            C(O)N(R⁷)R⁸ and, in the case of cycloalkyl or heterocyclyl            groups, oxo; wherein            -   R⁷, R⁸ and R⁹ are as defined above;    -   each of R² and R³ is independently C₁₋₁₀ alkyl, wherein one or        more —CH₂— groups is optionally replaced by —O—, —S— or —NR⁷—        provided that adjacent —CH₂— groups are not so replaced and        which is optionally substituted with one or more substituents        selected from halo, OH, SH, N(R⁷)R⁸, aryl, heteroaryl,        cycloalkyl, heterocyclyl, —C(O)OR⁷, —C(O)N(R⁷)R⁸, OR⁷ and        —N(R⁷)R⁸, wherein R⁷ and R⁸ are as defined above;    -   R⁴ is H, halo, cyano, C₁₋₆ alkyl, C(O)OR¹⁶ or C(O)N(R¹⁶)R¹⁷;    -   wherein alkyl groups are optionally substituted with one or more        substituents selected from halo, —OR⁷ and —N(R⁷)R⁸, wherein R⁷        and R⁸ are as defined above;    -   each R¹⁶ and R¹⁷ is independently H or C₁₋₆ alkyl or R¹⁶ and R¹⁷        together with the nitrogen atom to which they are attached may        form a 5- or 6-membered heterocyclic ring optionally containing        one or more further heteroatoms selected from O, N and S; and    -   R⁵ is H or methyl.

The compounds of general formula (I) have ENaC blocking activity and,furthermore, are expected to have one or both of the followingadvantageous properties.

-   -   Effective mucocilliary clearance in vivo.    -   Prolonged lung retention so reducing the dose required to give        adequate inhibition of ENaC commensurate with b.i.d. or q.d.        dosing without leading to hyperkalaemia.

DESCRIPTION

In the present specification, except where the context requiresotherwise due to express language or necessary implication, the word“comprises”, or variations such as “comprises” or “comprising” is usedin an inclusive sense i.e. to specify the presence of the statedfeatures but not to preclude the presence or addition of furtherfeatures in various embodiments of the invention.

In the present specification, references to “pharmaceutical use” referto use for administration to a human or an animal, in particular a humanor a mammal, for example a domesticated or livestock mammal, for thetreatment or prophylaxis of a disease or medical condition. The term“pharmaceutical composition” refers to a composition which is suitablefor pharmaceutical use and “pharmaceutically acceptable” refers to anagent which is suitable for use in a pharmaceutical composition. Othersimilar terms should be construed accordingly.

In the context of the present specification, the term “plurality” refersto two or more.

The anion X⁻ can have any negative charge and will be balanced by theappropriate number of cations. Thus, for example, a compound of generalformula (I) in which X⁻ is an anion having a single negative charge willhave a 1:1 ratio of cation:anion whereas if the anion X⁻ has a charge of−2, the ratio of cation:anion in the compound of general formula (I)will be 2:1. The anion X⁻ is suitably a pharmacologically acceptableanion, although other anions may also be useful, particularly insynthetic precursors to the compounds of general formula (I). Suitableanions, X⁻ include halide, sulfate, nitrate, phosphate, formate,acetate, trifluoroacetate, fumarate, citrate, tartrate, oxalate,succinate, mandelate, methane sulfonate and p-toluene sulfonate. Anadditional anion X⁻ or an anion with additional negative charge, e.g. acharge of −2, will be required if the R¹ substituent contains a moietyR¹⁰ which is cationic such that the charge in the compound of generalformula (I) is balanced.

All of the compounds of general formula (I) are salts. In the presentspecification, references to salts of the compounds of formula (I) mayrefer to salts of an additional basic nitrogen atom, for example anitrogen atom to which R⁷ and R⁸ moieties are attached. Counter ions forsuch salts are as defined for X⁻.

Alternatively, when R¹, R² or R³ comprises a carboxyl group C(O)OH,salts may be formed. Suitable counter ions for such salts includesodium, potassium, calcium, aluminium, zinc, magnesium and other metalions as well as choline, diethanolamine, ethanolamine, ethyl diamine,megulmine and other well-known basic addition salts as summarised inPaulekuhn et al., (2007) J. Med. Chem. 50: 6665-6672 and/or known tothose skilled in the art. In some cases, R² or R³ may comprise ananionic group, for example C(O)O⁻, which may act as counter ion to theN⁺ moiety in the benzimidazolium ring.

In the present specification, the term “C₁₋₆” alkyl refers to a straightor branched fully saturated hydrocarbon group having from 1 to 6 carbonatoms. The term encompasses methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl and t-butyl. Other alkyl groups, for example C₁₋₁₂ alkyl andC₁₋₄ alkyl are as defined above but contain different numbers of carbonatoms.

The term “C₂₋₆ alkenyl” refers to a straight or branched hydrocarbongroup having from 2 to 6 carbon atoms and at least one carbon-carbondouble bond. Examples include ethenyl, prop-1-enyl, hex-2-enyl etc.Other alkenyl groups, for example C₁₋₁₂ alkenyl are as defined aboveexcept that they contain the specified number (e.g. 1 to 12) carbonatoms.

The term “C₂₋₆ alkynyl” refers to a straight or branched hydrocarbongroup having from 2 to 6 carbon atoms and at least one carbon-carbontriple bond. Examples include ethynyl, prop-1-ynyl, hex-2-ynyl etc.Other alkynyl groups, for example C₂₋₁₂ alkynyl are as defined aboveexcept that they contain the specified number (e.g. 2 to 12) carbonatoms.

The term “C₁₋₆ alkylene” refers to a straight or branched fullysaturated hydrocarbon chain having from 1 to 6 carbon atoms. Examples ofalkylene groups include —CH₂—, —CH₂CH₂—, CH(CH₃)—CH₂—, CH₂CH(CH₃)—,—CH₂CH₂CH₂—, —CH₂CH(CH₂CH₃)— and —CH₂CH(CH₂CH₃)CH₂—. Other alkylenegroups, for example C₁₋₁₂ alkylene are as defined above except that theycontain the specified number (e.g. 1 to 12) carbon atoms.

The term “C₂₋₆ alkenylene” refers to a straight or branched hydrocarbonchain containing from 2 to 6 carbon atoms and at least one carbon-carbondouble bond. Examples of alkenylene groups include —CH═CH—, —CH═C(CH₃)—,—CH₂CH═CH—, —CH═CHCH₂—, CH₂CH₂CH═CH—, CH₂CH═C(CH₃)— and—CH₂CH═C(CH₂CH₃)—. Other alkenylene groups, for example C₂₋₁₂alkenylene, are as defined above except that they contain the specifiednumber (e.g. 2 to 12) carbon atoms.

The term “C₂₋₆ alkynylene” refers to a straight or branched hydrocarbonchain containing from 2 to 6 carbon atoms and at least one carbon-carbontriple bond. Examples of alkenylene groups include —C═C—, —CH₂C═C—,—C═C—CH₂—, CH₂CH₂C═C—, CH₂C═CHCH₂— and —CH₂CH═C—CH₂CH₂—)—. Otheralkynylene groups, for example C₂₋₁₂ alkynylene, are as defined aboveexcept that they contain the specified number (e.g. 2 to 12) carbonatoms.

The terms “carbocyclic” and “carbocyclyl” refer to a non-aromatichydrocarbon ring system containing from 3 to 10 ring carbon atoms,unless otherwise indicated, and optionally one or more double bond. Thecarbocyclic group may be a single ring or may contain two or three ringswhich may be fused or bridged. Examples include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclopentenyl and cyclohexenyl.

In the context of the present specification, the terms “heterocyclic”and “heterocyclyl” refer to a non-aromatic ring system containing 3 to10 ring atoms including at least one heteroatom selected from N, O andS. The heterocyclic group may be a single ring or may contain two orthree rings which may be fused or bridged. Examples includetetrahydrofuranyl, tetrahydroypranyl, pyrrolidine, piperidinyl,morpholinyl, piperazinyl and thiomorpholinyl.

The terms “aryl” and “aromatic” in the context of the presentspecification refer to a ring system with aromatic character having from5 to 14 ring carbon atoms and containing up to three rings. Where anaryl group contains more than one ring, not all rings must be fullyaromatic in character. Examples of aromatic moieties are benzene,naphthalene, fluorene, indane and indene.

The terms “heteroaryl” and “heteroaromatic” in the context of thespecification refer to a ring system with aromatic character having from5 to 14 ring atoms, at least one of which is a heteroatom selected fromN, O and S, and containing up to three rings. Where a heteroaryl groupcontains more than one ring, not all rings must be fully aromatic incharacter. Examples of heteroaryl groups include pyridine, pyrimidine,indole, benzofuran, benzimidazole and indolene.

The term “halogen” refers to fluorine, chlorine, bromine or iodine, theterm “halo” to fluoro, chloro, bromo or iodo groups and “halide” tofluoride, chloride, bromide or iodide.

The term “C₁₋₆ haloalkyl” as used herein refers to a C₁₋₆ alkyl group asdefined above in which one or more of the hydrogen atoms is replaced bya halo group. Any number of hydrogen atoms may be replaced, up toperhalo substitution. Examples include trifluoromethyl, chloroethyl and1,1-difluoroethyl. Other haloalkyl groups, for example C₁₋₁₂ haloalkylare as defined above except that they contain the specified number (e.g.1 to 12) carbon atoms.

The term “isotopic variant” refers to isotopically-labelled compoundswhich are identical to those recited in formula (I) but for the factthat one or more atoms are replaced by an atom having an atomic mass ormass number different from the atomic mass or mass number most commonlyfound in nature, or in which the proportion of an atom having an atomicmass or mass number found less commonly in nature has been increased(the latter concept being referred to as “isotopic enrichment”).Examples of isotopes that can be incorporated into compounds of theinvention include isotopes of hydrogen, carbon, nitrogen, oxygen,fluorine, iodine and chlorine such as 2H (deuterium), 3H, 11C, 13C, 14C,18F, 123I or 125I (e.g. 3H, 11C, 14C, 18F, 123I or 125I), which may benaturally occurring or non-naturally occurring isotopes.

The concept of canonical forms is well understood by the person of skillin the art. Thus, a compound of general formula (I) can have canonicalforms as follows:

Both of these canonical forms are included within the scope of theinvention.

The R¹ substituent is suitably at the 5- or the 6-position and thus thecompound of general formula (I) can be a compound of general formula(IA):

wherein R¹, R², R³, R⁴ R⁵ and X⁻ are as defined for general formula (I);or a compound of general formula (IB):

wherein R¹, R², R³, R⁴ R⁵ and X⁻ are as defined for general formula (I).

It should be noted that, because the compound of general formula (I) canhave different canonical forms as discussed above, if R² and R³ are thesame then the 5- and 6-positions are equivalent.

In some suitable compounds of general formula (I), R¹ is:

H, halo, —R¹², —C(O)OR¹² or —OR¹²; in particular, H, halo, or C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, —O(C₁₋₆ alkyl), —O(C₂₋₆ alkenyl) or—O(C₂₋₆ alkynyl), any of which is optionally substituted by one or morehalo substituents.

More suitably in these compounds, R¹ is H, halo, C₁₋₆ alkyl, C₁₋₆haloalkyl, C₁₋₆ alkoxy or C₁₋₆ haloalkoxy.

Examples of such R¹ groups include H, chloro, methyl, trifluoromethyl,methoxy and trifluoromethoxy.

In other suitable compounds of general formula (I), R¹ is -L¹R¹⁰.

Suitably in such compounds, L¹ is:

-   -   —Z¹—, -Q¹-, —Z¹Q¹-, -Q¹Z¹—, —Z¹Q¹Z²—, -Q¹Q²-, -Q¹Q²Z¹—,        -Q¹Q²Z¹Q³Z²—;    -   —OZ¹—, —OZ¹Q¹-, —OZ¹Q¹Z²—;    -   —Z¹N(R⁷)Z²—, -Q¹Z¹N(R⁷)Z²—;    -   —C(O)Q¹-, —C(O)Q¹Z¹—, —C(O)Q¹Q²-, —C(O)Q¹Q²Z¹—,        —C(O)Q¹N(R⁷)C(O)Z¹—, —C(O)Q¹N(R⁷)C(O)Z¹Q²-;    -   —C(O)N(R⁷)Z¹—, —C(O)N(R⁷)Q¹-, —C(O)N(R⁷)Z¹Q¹-,        —C(O)N(R⁷)Z¹Q¹Z²—, —C(O)N(R⁷)Q¹Z¹—, —C(O)N(R⁷)Q¹Q²-,        —C(O)N(R⁷)Q¹Q²Z¹—, —C(O)N(R⁷)Z¹Q¹Q²Z²—, —C(O)N(R⁷)Z¹O(CH₂CH₂O),        Z²—, —C(O)N(R⁷)Z¹O(CH₂O)_(n)Z²—, —C(O)N(R⁷)Z¹Q¹Z²N(R⁸)Z³—,        —C(O)N(R⁷)Z¹N(R⁸)Z²—, —C(O)N(R⁷)Q¹Z¹N(R⁸)Z²—,        —C(O)N(R⁷)Z¹Q¹OQ²OQ³-, —C(O)N(R⁷)Z¹Q¹OQ²OQ³Z²—;    -   —C(O)OZ¹—, —C(O)OZ¹Q¹-, —C(O)OZ¹Q¹Z²—;    -   Q¹C(O)Q²-, Q¹C(O)Z¹—, -Q¹C(O)Q²Z¹—, Q¹C(O)Q²Q³-, Q¹C(O)Z¹Q²- or        Q¹C(O)Q²Q³Z¹—.

In some more suitable compounds, L¹ is:

-   -   Z¹—, -Q¹-, -Q¹Z¹—, -Q¹Q²-, -Q¹Q²Z¹—, -Q¹Q²Z¹Q³Z²—;    -   —OZ¹—;    -   —Z¹N(R⁷)Z²—, -Q¹Z¹N(R⁷)Z²—;    -   —C(O)Q¹-, —C(O)Q¹Z¹—, —C(O)Q¹Q²-, —C(O)Q¹N(R⁷)C(O)Z¹—;    -   —C(O)N(R⁷)Z¹—, —C(O)N(R⁷)Q¹-, —C(O)N(R⁷)Z¹Q¹-,        —C(O)N(R⁷)Z¹Q¹Z²—, —C(O)N(R⁷)Q¹Z¹—, —C(O)N(R⁷)Q¹Q²-,        —C(O)N(R⁷)Q¹Q²Z¹—, —C(O)N(R⁷)Z¹Q¹Q²Z²—,        —C(O)N(R⁷)Z¹O(CH₂CH₂O)_(n)Z²—, —C(O)N(R⁷)Z¹N(R⁸)Z²—,        —C(O)N(R⁷)Z¹Q¹OQ²OQ³Z²—; or    -   Q¹C(O)Q²-.

In other more suitable compounds, L¹ is:

-   -   Z¹—, -Q¹-, -Q¹Z¹—, -Q¹Q²-, -Q¹Q²Z¹—;    -   —OZ¹—;    -   —C(O)Q¹-, —C(O)Q¹Z¹—;    -   —C(O)N(R⁷)Z¹—, —C(O)N(R⁷)Q¹-, —C(O)N(R⁷)Z¹Q¹-, —C(O)N(R⁷)Q¹Z¹—,        —C(O)N(R⁷)Z¹Q¹Q²Z²—, —C(O)N(R⁷)Z¹O(CH₂CH₂O)_(n)Z²— or        —C(O)N(R⁷)Z¹Q¹Z²N(R⁸)Z³—.

In some suitable compounds where R¹ is -LR¹⁰, the cyclic groups Q¹, Q²and Q³ are independently selected from 5- and 6-membered aryl andheteroaryl groups and 4 to 8-membered carbocyclyl and heterocyclylgroups.

More suitably, Q¹, Q² and Q³ are selected from phenyl, 5- and 6-memberedheteroaryl groups and 4- to 7-membered and heterocyclyl groups, stillmore suitably phenyl, 5- and 6-membered nitrogen-containing heteroaryland 4- to 7-membered nitrogen-containing heterocyclyl groups.

Examples of such heteroaryl Q¹, Q² and Q³ groups include pyridyl,pyrimidinyl, pyrazolyl, imidazolyl and oxazolyl groups, with 5-memberedrings such as imidazolyl and oxazolyl and especially pyrazolyl beingparticularly suitable. When Q¹, Q² or Q³ is pyrazolyl, it may have thefollowing regiochemistry:

Where a and b show the links to the remainder of the molecule.

Examples of heterocyclyl Q¹, Q² and Q³ groups include azetidinyl,piperidinyl, piperazinyl, and aziridinyl, with 6-membered rings such aspiperazinyl and piperidinyl being more suitable. Piperidinyl is aparticularly suitable heterocyclyl group, especially 1,4-piperidinyl.

Other more suitable Q¹, Q² and Q³ groups include cyclohexyl andtetrohydropyran groups, either or which may be substituted with one ormore substituents selected from OH, or NR¹⁵R¹⁶, especially OH, NH₂ orNHCH₃.

When L¹ comprises a C(O) moiety linked to a Q moiety, the Q moiety maybe a nitrogen containing heterocyclyl ring in which C(O) is linked tothe nitrogen atom.

For example, in —C(O)Q¹-, —C(O)Q¹Z¹—, —C(O)Q¹Q²-, —C(O)Q¹Q²Z¹—,—C(O)Q¹N(R⁷)C(O)Z¹—, —C(O)Q¹N(R⁷)C(O)Z¹Q²-, —C(O)Q¹N(R⁷)C(O)Z¹Q²Q³-,—C(O)Q¹N(R⁷)C(O)Z¹Q²Z²—, Q¹ is suitably a 5- or 6-membered heterocyclylring which is linked to the —C(O) moiety via a ring nitrogen atom.Suitably, Q¹ is a 5- or 6-membered nitrogen-containing heterocyclyl ringsuch as piperidin-1-yl or pyrrolidine-1-yl, more suitably piperidin-1yl.Suitably, when Q¹ is piperidin-1-yl the remainder of the molecule islinked to the 4-position of the piperidine ring. When Q¹ ispyrrolidine-1-yl, the remainder of the molecule may be linked to the3-position of the pyrrolidine ring.

When L¹ is -Q¹C(O)Q²-, -Q¹C(O)Q²Z¹—, Q¹C(O)Q²Q³-, Q¹C(O)Q²Q³Z¹—, Q² issuitably a 5- or 6-membered heterocyclyl ring which is linked to the—C(O) moiety via a ring nitrogen atom. Suitably, Q² is piperidin-1-yl orpyrrolidine-1-yl, more suitably piperidin-1yl. Suitably, when Q¹ ispiperidin-1-yl the remainder of the molecule is linked to the 4-positionof the piperidine ring. When Q¹ is pyrrolidine-1-yl, the remainder ofthe molecule may be linked to the 3-position of the pyrrolidine ring.

In other cases, however, when L¹ comprises a C(O) moiety linked to a Qmoiety, the Q moiety may be a heterocyclyl group which is linked to theC(O) moiety via a ring carbon atom. Examples of heterocyclyl groupsinclude 5- and 6-membered rings, suitably nitrogen-containing rings suchas piperidinyl or pyrrolidinyl. Suitably, in this case, when Q¹ is apiperidine ring it is a piperidin-4-yl group such that the piperidine4-position is linked to the C(O) moiety. Suitably, the piperidinenitrogen atom is linked to the remainder of the molecule. Examples of L¹moieties of this type include C(O)Q¹Z¹, in which Q¹ may be a piperidinering in which the 4-position is linked to C(O) and the 1-position islinked to Z¹.

When L¹ comprises a —C(O)N(R⁷)— moiety linked to a Q moiety, the Qmoiety is suitably a heterocyclyl ring, e.g. a 5- or 6-memberednitrogen-containing heterocyclyl ring, which is linked to the—C(O)N(R⁷)— moiety via a ring carbon atom.

For example, when L¹ is —C(O)N(R⁷)Q¹-, —C(O)N(R⁷)Q¹Z¹—, —C(O)N(R⁷)Q¹Q²-,—C(O)N(R⁷)Q¹Q²Z¹—, —C(O)N(R⁷)Q¹Z¹N(R⁸)Z²—, —Z¹C(O)N(R⁷)Q¹-,—Z¹C(O)N(R⁷)Q¹Z²— or —Z¹C(O)N(R⁷)Q¹Q²-, Q¹ is suitably a 5- or6-membered heterocyclyl ring which is linked to the —C(O)N(R⁷)— moietyvia a ring carbon atom, with the remainder of the molecule being linkedto a ring nitrogen atom. Suitably, Q¹ is piperidin-4-yl or pyrrolidinyl.When Q¹ is piperidin-4-yl, the remainder of the molecule is suitablylinked to the 1-position of the piperidine ring.

For other L¹ groups in which Q¹ and/or Q² and/or Q² is piperidinyl, theyare suitably either piperidin-1-yl or piperidin-4-yl.

When the L¹ comprises a -Q¹Q²- or -Q²Q³-moiety, this may be, forexample:

Where a and b show the links to the remainder of the molecule.

Other examples of -Q¹Q²- and -Q²Q³-moieties include biphenyl, suitably a1,1′-biphenyl-4-yl moiety. This type of -Q¹Q²- or -Q²Q³-moiety issuitably linked at each side to a Z moiety, for example as in—C(O)N(R⁷)Z¹Q¹Q²Z²—

In some suitable compounds where R¹ is -L¹R¹⁰, and L¹ contains Z² andoptionally Z² and Z³, each of Z¹, Z² and Z³ is independently C₁₋₁₂alkylene, optionally substituted by one or more halo or OH groups.

In some compounds of the invention, R¹ is L¹R¹⁰ and L¹ comprises a Z¹,and optionally a Z² and optionally a Z³ moiety.

In compounds where L¹ is:

-   -   —Z¹—, —Z¹Q¹-, -Q¹Z¹—, —Z¹Q¹Z²—, -Q¹Q²Z¹—, -Q¹Q²Z¹Q³Z²—,        —Z¹Q¹OQ²OQ³-;    -   —OZ¹—, —OZ¹Q¹-, —OQ¹Z¹—, —OZ¹Q¹Z²—, —OQ¹Q²Z¹—, —OQ¹Q²Z¹Q³Z²—,        —OZ¹Q¹OQ²OQ³-;    -   —Z¹N(R⁷)Z²—, -Q¹ZN(R⁷)Z²—, —Z¹N(R⁷)Z²Q¹-, -Q¹ZN(R⁷)Z²Q²Z³—;    -   —Z¹O(CH₂CH₂O)_(n)Z²—, —Z¹O(CH₂CH₂O)_(n)Q¹-,        —Z¹O(CH₂CH₂O)_(n)Z²Q¹, —Z¹O(CH₂CH₂O)_(n)Q¹Z²—,        -Q¹Z¹O(CH₂CH₂O)_(n)Z²—, -Q¹Z¹O(CH₂CH₂O)_(n)Q¹-,        -Q¹Z¹O(CH₂CH₂O)_(n)Z²Q¹, —Z¹O(CH₂CH₂O)_(n)Z²Q¹Z³—;    -   —C(O)Z¹—, —C(O)Z¹Q¹-, —C(O)Z¹Q¹Z²—, —C(O)Q¹Z¹—, —C(O)Q¹Q²Z¹—,        —C(O)Q¹N(R⁷)C(O)Z¹—, —C(O)Q¹N(R⁷)C(O)Z¹Q²-,        —C(O)Q¹N(R⁷)C(O)Z¹Q²Q³-—C(O)Q¹N(R⁷)C(O)Z¹Q²Z²—,        —C(O)Z¹Q¹OQ²OQ³-;    -   —C(O)N(R⁷)Z¹—, —C(O)N(R⁷)Z¹Q¹-, —C(O)N(R⁷)Z¹Q¹Z²—,        —C(O)N(R⁷)Q¹Z¹—, —C(O)N(R⁷)Q¹Q²Z¹—, —C(O)N(R⁷)Z¹Q¹Q²Z²—,        —C(O)N(R⁷)Z¹O(CH₂CH₂O)_(n)Z²—, —C(O)N(R⁷)Z¹O(CH₂O)_(n)Z²—,        —C(O)N(R⁷)Z¹Q¹Z²N(R⁸)Z³—, —C(O)N(R⁷)Z¹N(R⁸)Z²—,        —C(O)N(R⁷)Q¹Z¹N(R⁸)Z²—, —C(O)N(R⁷)Z¹Q¹OQ²OQ³-,        —C(O)N(R⁷)Z¹Q¹OQ²OQ³Z²—;    -   Z¹C(O)N(R⁷)Z²—, —Z¹C(O)N(R⁷)Q¹-, —Z¹C(O)N(R⁷)Z²Q¹-,        —Z¹C(O)N(R⁷)Q¹Z²—, —Z¹C(O)N(R⁷)Q¹Q²-,    -   —Z¹C(O)Q¹-, —Z¹C(O)Q¹Z²—, —Z¹C(O)Q¹Q²-, —Z¹C(O)N(R⁷)Q¹Q²Z²—;    -   —C(O)OZ¹—, —C(O)OZ¹Q¹-, —C(O)OZ¹Q¹Z²—, —C(O)OQ¹Z¹—, —C(O)OQ¹Q²-,        —C(O)OQ¹Q²Z¹—;    -   Q¹C(O)Z¹—, -Q¹C(O)Q²Z¹—, -Q¹C(O)Z¹Q²-, Q¹C(O)Q²Q³Z¹—;    -   —C(═NR⁹)N(R⁷)Z¹—, —C(═NR⁹)N(R⁷)Q¹-, —C(═NR⁹)N(R⁷)Z¹Q¹-,        —C(═NR⁹)N(R⁷)Z¹Q¹Z²—, —C(═NR⁹)N(R⁷)Q¹Z¹—, —C(═NR⁹)N(R⁷)Q¹Q²-,        —C(═NR⁹)N(R⁷)Q¹Q²Z¹—;    -   the groups Z¹ and, where present, Z² and Z³ suitably comprise        C₁₋₃ alkylene, C₂₋₈ alkenylene, C₂₋₈ alkynylene any of which is        optionally substituted by one or more halo, OH, C(O)NR¹⁵R¹⁶,        C(O)OR¹⁵ or NR¹⁵R¹⁶; wherein R¹⁵ and R¹⁶ are as defined above.

In some more suitable compounds, the groups Z¹ and, where present, Z²and Z³ comprise C₁₋₆ alkylene optionally substituted by one or morehalo, OH, C(O)NR¹⁵R¹⁶, C(O)OR¹⁵ or NR¹⁵R¹⁶; wherein R¹⁵ and R¹⁶ are asdefined above.

Still more suitably, the groups Z¹ and, where present, Z² and Z³comprise C₁₋₄ alkylene which is unsubstituted or substituted by one ormore halo, OH, C(O)NR¹⁵R¹⁶, C(O)OR¹⁵ or NR¹⁵R¹⁶, wherein each R¹⁵ andR¹⁶ is H. Typically, Z¹ and where present, Z² and Z³ comprise C₁₋₄alkylene which is unsubstituted or substituted with one or more OH,halo, C(O)NH₂ or C(O)OH.

In some particularly suitable compounds, Z¹ and, where present, Z² andZ³ are unsubstituted C₁₋₄ alkylene.

In other particularly suitable compounds Z¹ is C₁₋₄ alkylene substitutedwith one or more halo, C(O)NH₂ or C(O)OH.

These Z¹, Z² and Z³ moieties are particularly suitable when L¹ is:

-   -   —Z¹—, -Q¹Z¹— where Q¹ is linked via a ring carbon atom to Z¹,        -Q¹Q²Z¹— where Q² is linked via a ring carbon atom to Z¹;    -   —OZ¹—;    -   —C(O)Q¹Z¹—, where Q¹ is linked via a ring nitrogen atom to C(O)        and via a ring carbon atom to Z¹;    -   —C(O)N(R⁷)Z¹—, —C(O)N(R⁷)Q¹Z¹—, —C(O)N(R⁷)Z¹Q¹Q²Z²—,        —C(O)N(R⁷)Z¹O(CH₂CH₂O)_(n)Z²—, —C(O)N(R⁷)Z¹Q¹Z²N(R⁸)Z³—.

In some compounds of general formula (I), Z¹, Z² or Z³ may be directlylinked to a cyclic group via a ring nitrogen atom. This may occur, forexample, in compounds where R¹ is L¹R¹⁰ and L¹ is:

-   -   -Q¹Z¹—, —Z¹Q¹Z²—, -Q¹Q²Z¹—, —OQ¹Z¹—, —OZ¹Q¹Z²—, —OQ¹Q²Z¹—,        —Z¹O(CH₂CH₂O)_(n)Q¹Z²—, -Q¹Z¹O(CH₂CH₂O)_(n)Z²—,        —Z¹O(CH₂CH₂O)_(n)Z²Q¹Z³—, —C(O)Z¹Q¹Z²—, —C(O)Q¹Z¹—,        —C(O)Q¹Q²Z¹—, —C(O)Q¹N(R⁷)C(O)Z¹Q²Z²—, —C(O)N(R⁷)Z¹Q¹Z²—,        —C(O)N(R⁷)Q¹Z¹—, —C(O)N(R⁷)Q¹Q²Z¹—, —C(O)N(R⁷)Z¹Q¹Q²Z²—,        —C(O)N(R⁷)Z¹Q¹OQ²OQ³Z²—, Z¹C(O)N(R⁷)Q¹Z²—, —Z¹C(O)Q¹Z²—,        Z¹C(O)N(R⁷)Q¹Q²Z²—, —C(O)OZ¹Q¹Z²—, —C(O)OQ¹Z¹—, —C(O)OQ¹Q²Z¹—;        Q¹C(O)Q²Z¹—, Q¹C(O)Q²Q³Z¹—, —C(═NR⁹)N(R⁷)Z¹Q¹Z²—,        —C(═NR⁹)N(R⁷)Q¹Z¹—, —C(═NR⁹)N(R⁷)Q¹Q²Z¹—.

In some such compounds, the Z¹ or Z² or Z³ group is also linked to R¹⁰.Where this is the case, the Z¹ or Z² or Z³ group may be C₁₋₁₂ alkylenesubstituted with one or more OH group, suitably by a plurality of OHgroups, for example 2-11 OH groups. Typically, the number of OH groupswill be one less than the number of carbon atoms in the alkylene group;More suitably in this case, Z¹ is a C₁₋₈ alkylene group substituted with2-7 OH groups, for example 5-7 OH groups.

Examples of suitable Z¹ or Z² or Z³ groups of this type include—CH₂[CH(OH)_(n)]—, where n is suitably 3-7. Most suitably, Z¹ or Z² orZ³ where appropriate is —CH₂—CH(OH)—CH(OH)—CH(OH)—CH(OH)—CH(OH)—.

In such compounds, R¹⁰ is suitably H such that the Z¹R¹⁰, Z²R¹⁰ or Z³R¹⁰moiety is —CH₂[CH(OH)]_(n)—H, where n is suitably 3-7, for example—CH₂—[CH(OH)]₄—CH₂OH.

In compounds where R¹ is -L¹R¹⁰, suitable R¹⁰ groups include H,—N(R⁷)R⁸, —N(R⁷)C(═NR⁹)N(R⁸)₂, —N(R⁷)C(O)OR⁸, —N(R⁷)—C(O)—(C₁₋₃alkylene)-N⁺(R⁸)₃, —N⁺(R⁸)₃, OR⁷ or —C(O)OR⁷.

More suitably, R¹⁰ is H, —N(R⁷)R⁸, —N(R⁷)C(═NR⁹)N(R⁸)₂, —N(R⁷)C(O)OR⁸ or—C(O)OR⁷.

When R¹⁰ is H, L¹ is suitably —Z¹—, Q¹, -Q¹Z¹—, —Z¹Q¹Z²—, -Q¹Q²-Q¹Q²Z¹—;

-   -   —OZ¹—, —OQ¹Z¹—, —OZ¹Q¹Z²—, —OQ¹Q²Z¹—;    -   —Z¹O(CH₂CH₂O)_(n)Q¹Z²—, -Q¹Z¹O(CH₂CH₂O)_(n)Z²—,        —Z¹O(CH₂CH₂O)_(n)Z²Q¹Z³—,    -   —C(O)Z¹—, —C(O)Z¹Q¹Z²—, —C(O)Q¹Z¹—, —C(O)Q¹Q²Z¹—;    -   —C(O)N(R⁷)Z¹—, —C(O)N(R⁷)Q¹-, —C(O)Q¹N(R⁷)C(O)Z¹Q²Z²—,        —C(O)N(R⁷)Z¹Q¹Z²—, —C(O)N(R⁷)Q¹Z¹—, —C(O)N(R⁷)Q¹Q²Z¹—,        —C(O)N(R⁷)Z¹Q¹Q²Z²—, —C(O)N(R⁷)Z¹Q¹OQ²OQ³Z²—, Z¹C(O)N(R⁷)Q¹Z²—;    -   —C(O)N(R⁷)Q¹Q²Z¹—, —Z¹C(O)Q¹Z²—, Z¹C(O)N(R⁷)Q¹Q²Z²—;    -   —C(O)OZ¹—, —C(O)OZ¹Q¹Z²—, —C(O)OQ¹Z¹—, —C(O)OQ¹Q²-,        —C(O)OQ¹Q²Z¹—;    -   —C(O)OQ¹Z¹—, —C(O)OQ¹Q²Z¹—; Q¹C(O)Q²Z¹—, Q¹C(O)Q²Q³Z¹—,        —C(═NR⁹)N(R⁷)Z¹—, C(═NR⁹)N(R⁷)Z¹Q¹Z²—, —C(═NR⁹)N(R⁷)Q¹Z¹—,        C(═NR⁹)N(R⁷)Q¹Q²Z¹—.

In some more suitable compounds, when R¹⁰ is H, L¹ is —OZ¹, where Z¹ isas defined above but is suitably C₁₋₈ alkylene optionally substituted asdescribed above. More suitably in these compounds, Z¹ is C₁₋₄ alkylenesuch that the group —OZ¹R¹⁰ is —O(C₁₋₄ alkyl), for example methoxy,ethyoxy isopropoxy or t-butyloxy. In one embodiment (e.g. as used inExamples 4, 7-14 and 19), R¹ is methoxy.

In other more suitable compounds, when R¹⁰ is H, L¹ is -Q¹-, -Q¹Q²- or—C(O)N(R⁷)Q¹-, where the Q¹ group or, for-Q¹Q²-, the Q² group, is anitrogen-containing heterocyclyl group which is linked to the R¹⁰ groupvia a ring nitrogen atom. Examples of such groups Q¹R¹⁰ or Q²R¹⁰ groupsinclude:

where * indicates the point at which the group is joined to theremainder of the molecule.

In an embodiment (e.g. as used in Example 30), R¹ is -Q¹-H.

In an embodiment (e.g. as used in Example 31), R¹ is -Q¹Q²-H.

In an embodiment (e.g. as used in Example 40), R¹ is —C(O)N(R⁷)Q¹-H.

In still other more suitable compounds, when R¹⁰ is H, L¹ is:

-   -   —Z¹—, Q¹, -Q¹Z¹—, -Q¹Q², -Q¹Q²Z¹—;    -   —OZ¹—, —OQ¹Z¹—, —OQ¹Q²Z¹—;    -   —C(O)Z¹—, —C(O)Q¹Z¹—, —C(O)Q¹Q²Z¹—;    -   —C(O)N(R⁷)Z¹—, —C(O)N(R⁷)Q¹Z¹—, —C(O)N(R⁷)Q¹Q²Z¹—;    -   —C(O)OZ¹—, —C(O)OQ¹Z¹—, —C(O)OQ¹Q²-, C(O)OQ¹Q²Z¹—;    -   —C(═NR⁹)N(R⁷)Z¹—, —C(═NR⁹)N(R⁷)Q¹Z¹— or C(═NR⁹)N(R⁷)Q¹Q²Z¹—.

In still other more suitable compounds, when R¹⁰ is H, and which containa cyclic group Q¹, Q² or Q³ linked to Z¹ or Z² or Z³, the cyclic groupmay be a nitrogen containing heterocyclyl group linked to Z¹ or Z² or Z³via a ring nitrogen atom. This may occur, for example, in compoundswhere R¹ is L¹R¹⁰ and L¹ is:

-   -   -Q¹Z¹—, —Z¹Q¹Z²—, -Q¹Q²Z¹—;    -   —OQ¹Z¹—, —OZ¹Q¹Z²—, —OQ¹Q²Z¹—;    -   —Z¹O(CH₂CH₂O)_(n)Q¹Z²—, -Q¹Z¹O(CH₂CH₂O)_(n)Z²—,        —Z¹O(CH₂CH₂O)_(n)Z²Q¹Z³—;    -   —C(O)Z¹Q¹Z²—, —C(O)Q¹Z¹—, —C(O)Q¹Q²Z¹—, —C(O)Q¹N(R⁷)C(O)Z¹Q²Z²—,        —C(O)N(R⁷)Z¹Q¹Z²—, —C(O)N(R⁷)Q¹Z¹—;    -   —C(O)N(R⁷)Q¹Q²Z¹—, —C(O)N(R⁷)Z¹Q¹Q²Z²—, —C(O)N(R⁷)Z¹Q¹OQ²OQ³Z²—;    -   Z¹C(O)N(R⁷)Q¹Z²—, —Z¹C(O)Q¹Z²—, Z¹C(O)N(R⁷)⁷Q¹Q²Z²—;    -   —C(O)OZ¹Q¹Z²—, —C(O)OQ¹Z¹—, —C(O)OQ¹Q²Z¹—;    -   Q¹C(O)Q²Z¹—, Q¹C(O)Q²Q³Z¹—;    -   —C(═NR⁹)N(R⁷)Z¹Q¹Z²—, —C(═NR⁹)N(R⁷)Q¹Z¹— or        —C(═NR⁹)N(R⁷)Q¹Q²Z¹—.

Particularly suitable compounds of this type are those in which R¹⁰ is Hand L¹ is -Q¹Z¹—, -Q¹Q²Z¹—, —C(O)N(R⁷)Q¹Z¹—, or —C(O)Q¹Z¹—; or, stillmore suitably, -Q¹Z¹—, -Q¹Q²Z¹— or —C(O)N(R⁷)Q¹Z¹—.

In compounds where R¹⁰ is H and in which L¹ contains a moiety Z¹, Z² orZ³ linked directly to R¹⁰, the Z¹, Z² or Z³ moiety is suitably a C₁₋₁₂alkylene group substituted by a plurality of OH groups, for example 2 to11 OH groups. Typically, the number of OH groups will be one less thanthe number of carbon atoms in the alkylene group;

More suitably in this case, Z¹ is a C₁₋₈ alkylene group substituted with2-7 OH groups, for example 5-7 OH groups.

Examples of suitable Z¹ groups of this type include —CH₂[CH(OH)]_(n)—,where n is suitably 3-7. Most suitably, n is 5 and in this case, Z¹ is—CH₂[CH(OH)]₄—CH(OH)— such that the group Z¹R¹⁰, Z²R¹⁰ or Z³R¹⁰ is amoiety —CH₂[CH(OH)]₄—CH₂OH.

Examples of compounds where the group Z¹R¹⁰ is a moiety—CH₂[CH(OH)]₄—CH₂OH include those in which R¹ is:

-   -   -piperidinyl-4-yl-CH₂—CH(OH)—CH(OH)—CH(OH)—CH(OH)—CH₂(OH).    -   -pyrazol-4-yl-piperidin-4-yl-CH₂—CH(OH)—CH(OH)—CH(OH)—CH(OH)—CH₂(OH).    -   —C(O)NH-piperidin-4-yl-CH₂—CH(OH)—CH(OH)—CH(OH)—CH(OH)—CH₂(OH).

In an embodiment (e.g. as used in Example 46), R¹ is -Q¹Z¹—H.

In an embodiment (e.g. as used in Example 47), R¹ is -Q¹Q²Z¹—H.

In an embodiment (e.g. as used in Example 49), R¹ is C(O)N(R⁷)Q¹Z¹—H.

Other suitable compounds in which R¹⁰ is H include those in which R¹⁰ isattached to a ring nitrogen atom of a moiety Q¹, Q² or Q³ or the L¹group.

When R¹⁰ is other than H, Z¹, Z² and Z³, where present, may be—(CH₂)_(n)— where n is 1 to 6 or —O(CH₂)_(m)—, where m is 1 to 5.

In compounds where R¹⁰ is —C(O)OR⁷, L¹ is suitably:

-   -   -Q¹- or -Q¹Q²- where Q¹ or, for -Q¹Q²-, Q² is a carbocyclyl or        heterocyclyl group and is linked to R¹⁰ via a ring carbon atom;        or    -   C(O)N(R⁷)Q¹, where Q¹ is a is a carbocyclyl or heterocyclyl        group and is linked to R¹⁰ via a ring nitrogen atom.

In these compounds, R⁷ is suitably C₁₋₆ alkyl, still more suitably C₁₋₄alkyl, for example t-butyl.

In an embodiment (e.g. as used in Example 25), R¹ is -Q¹C(O)OR⁷, whereQ¹ is piperidin-4-yl and R⁷ is t-butyl.

In an embodiment (e.g. as used in Example 26), R¹ is -Q¹Q²C(O)OR⁷, whereQ¹ is pyrazol-4-yl, Q² is piperidin-4-yl and R⁷ is t-butyl.

In an embodiment (e.g. as used in Example 36), R¹ is C(O)NHQ¹C(O)OR⁷,where Q¹ is piperidin-1-yl and R⁷ is

When R¹⁰ is —N(R⁷)R⁸, —N(R⁷)C(═NR⁹)N(R⁸)₂ or —N(R⁷)C(O)OR⁸—, L¹ issuitably:

-   -   —Z¹—,    -   —OZ¹—;    -   —C(O)N(R⁷)Z¹—, —C(O)N(R⁷)Z¹Q¹Q²Z²—, —C(O)N(R⁷)Z¹Q¹Z²N(R⁸)Z³,        —C(O)N(R⁷)Z¹O(CH₂CH₂O)_(n)Z²;    -   —C(O)N(R⁷)Q¹-, —C(O)N(R⁷)Z¹Q¹- or —C(O)Q¹-, where Q¹ is a        carbocyclyl or heterocyclyl group and is linked to R¹⁰ via a        ring carbon atom; or C(O)Q¹Z¹—.

Compounds in which R¹⁰ is —N(R⁷)R⁸ are particularly suitable.

Typically, when R¹⁰ is —N(R⁷)R⁸, each of R⁷ and R⁸ is independentlyeither H or C₁₋₈ alkyl optionally substituted with one or more OHgroups. In some cases, both R⁷ and R⁸ are H.

In the case where either or both of R⁷ and R⁸ is C₁₋₃ alkyl, it may besubstituted with a plurality of OH groups, for example 2-7 OH groups.Typically, the number of OH groups will be one less than the number ofcarbon atoms in the alkyl group. More suitably in compounds of thistype, one or preferably both, of R⁷ and R⁸ may be —CH₂[CH(OH)]_(m)CH₂OH,where m is suitably 2-6. Most suitably, m is 4 and in this case, the R⁷and/or R⁸ group is a moiety —CH₂[CH(OH)]₄—CH₂OH.

In particularly suitable compounds R¹⁰ is —N{CH₂[CH(OH)]₄—CH₂OH}₂.

In an embodiment (e.g. as used in Example 29), L¹ is —Z¹— and R¹⁰ is NH₂

In an embodiment (e.g. as used in Examples 27 and 28), L¹ is —OZ¹— andR¹⁰ is NH₂.

In an embodiment (e.g. as used in Example 39), L¹ is —C(O)N(R⁷)Z¹— andR¹⁰ is NH₂.

In an embodiment (e.g. as used in Example 41), L¹ is —C(O)Q¹ and R¹⁰ isNH₂.

In an embodiment (e.g. as used in Example 42), L¹ is —C(O)N(R⁷)Z¹Q¹ andR¹⁰ is NH₂.

In an embodiment (e.g. as used in Examples 43 and 44), L¹ is —OZ¹— andR¹⁰ is —N{CH₂[CH(OH)]₄—CH₂OH}₂.

In an embodiment (e.g. as used in Example 45), L¹ is —Z¹— and R¹⁰ is—N{CH₂[CH(OH)]₄—CH₂OH}₂.

In an embodiment (e.g. as used in Examples 48, 57, 58 and 61), L¹ is—C(O)N(R⁷)Z¹— and R¹⁰ is —N{CH₂[CH(OH)]₄—CH₂OH}₂.

In an embodiment (e.g. as used in Examples 50, 53, 54, 65 and 66), L¹ is—C(O)Q¹ and R¹⁰ is —N{CH₂[CH(OH)]₄—CH₂OH}₂.

In an embodiment (e.g. as used in Example 51), L¹ is —C(O)N(R⁷)Z¹Q¹ andR¹⁰ is —N{CH₂[CH(OH)]₄—CH₂OH}₂.

In an embodiment (e.g. as used in Examples 55 and 56), L¹ is—C(O)N(R⁷)Q¹ and R¹⁰ is —N{CH₂[CH(OH)]₄—CH₂OH}₂.

In an embodiment (e.g. as used in Example 59), L¹ is—C(O)N(R⁷)Z¹O(CH₂CH₂O)_(n)Z²— and R¹⁰ is —N{CH₂[CH(OH)]₄—CH₂OH}₂.

In an embodiment (e.g. as used in Example 60), L¹ is —C(O)N(R⁷)Z¹Q¹Q²Z²—and R¹⁰ is —N{CH₂[CH(OH)]₄—CH₂OH}₂.

In an embodiment (e.g. as used in Example 62), L¹ is—C(O)N(R⁷)Z¹Q¹Z²N(R⁸)Z³— and R¹⁰ is —N{CH₂[CH(OH)]₄—CH₂OH}₂.

When R¹⁰ is —N(R⁷)C(═NR⁹)N(R⁸)₂, each of R⁷ and R⁹ is suitably H or C₁₋₄alkyl, particularly H or methyl and especially H; and each R⁸ isindependently either H or C₁₋₈ alkyl optionally substituted with one ormore OH groups. In the case where either or both R⁸ groups is a C₁₋₈alkyl group, it may be substituted with a plurality of OH groups, forexample 2-7 OH groups. Typically, the number of OH groups will be oneless than the number of carbon atoms in the alkyl group. More suitablyin compounds of this type, one or preferably both, R⁸ groups may be—CH₂[CH(OH)]_(m)CH₂OH, where m is suitably 2-6. Most suitably, m is 4and in this case, one or preferably both the R⁸ groups is a moiety—CH₂[CH(OH)]₄—CH₂OH.

In particularly suitable compounds —N(R⁷)C(═NR⁹)N(R⁸)₂ is:—NHC(═NH)—N{CH₂[CH(OH)]₄—CH₂OH}₂.

When R¹⁰ is —N(R⁷)C(O)OR⁸, each of R⁷ and R⁸ is suitably H or C₁₋₆alkyl. More suitably, R⁷ is H and R⁸ is C₁₋₆ alkyl, still more suitablyC₁₋₄ alkyl, for example t-butyl.

In still other compounds of general formula (I), R¹ is —OR¹² —SO₂R¹²,—C(O)OR¹², —C(O)NR¹²R¹³, —C(═NR⁹)NR¹²R¹³ -Q¹OR¹² -Q¹SO₂R¹², -Q¹C(O)OR¹²,-Q¹C(O)NR¹²R¹³, -Q¹C(═NR⁷)NR¹²R¹⁴, -Q¹Q²OR¹², -Q¹SO₂R¹², -Q¹Q²C(O)OR¹²,-Q¹Q²C(O)NR¹²R¹³ or -Q¹Q²C(═NR⁹)NR¹²R¹³;

Suitable groups Q¹ and Q² are as set out above.

Suitable R¹² and R¹³ groups include H and C₁₋₆ alkyl optionallysubstituted with one or more substituents selected from halo and OR⁷.

As mentioned above, each of R² and R³ is independently C₁₋₁₀ alkyl inwhich one or more —CH₂— groups is optionally replaced by —O—, —S— or—NR⁷— and which is optionally substituted as defined above. There may,for example, be no such substituents or a single substituent.

Suitably, each of R² and R³ is independently C₁₋₁₀ alkyl in which one ormore —CH₂— groups is optionally replaced by —O— or —S— and which isoptionally substituted as defined above.

Examples of R² and R³ groups include —(CH₂)_(s)CH₃ or (CH₂CH₂O)_(t)—H,either of which is optionally substituted as defined above; and whereins is 0-9, more suitably 0-6 and still more suitably 0-3; and t is 1-3,especially 2 or 3.

Particularly suitable compounds of general formula (I) are those inwhich R² and R³ are the same or different and are both unsubstitutedC₁₋₄ alkyl, for example methyl or ethyl. In some such compounds R² andR³ are the same and are both methyl or both ethyl. In other suchcompounds, one of R² and R³ is methyl and the other of R² and R³ isethyl.

Suitable substituents for R² and R³ include OH, SH, halo, N(R⁷)R⁸,C(O)OR⁷, C(O)N(R⁷)R⁸, phenyl or pyridyl, where R⁷ and R⁸ are as definedabove. Particularly suitable substituents for R² include OH, SH, phenylor pyridyl, particularly OH, phenyl, pyridyl, C(O)O—C₁₋₆ alkyl, C(O)OH,C(O)NH₂ or C(O)N(R⁷)R⁸, where each of R⁷ and R⁸ is C₁₋₃ alkyl or R⁷ andR⁸ together with the nitrogen atom to which they are attached form apyrrolidine, piperidine, piperazine or morpholine ring.

In some compounds of general formula (I), at least one of R² and R³ is—(CH₂)_(s)CH₃, wherein s is as defined above, and is optionallysubstituted with a single substituent as defined above.

In some such compounds of general formula (I) at least one of R² and R³is methyl, ethyl, benzyl, pyridylmethyl, —CH₂OH, —CH₂NH₂, —CH₂CH₂OH orCH₂CH₂NH₂.

In other particularly suitable compounds, at least one of R² and R³ is—CH₂CH₂OCH₂CH₂OH or —CH₂CH₂OCH₂CH₂OCH₂CH₂OH

In suitable compounds of general formula (I), R⁴ is H, halo, cyano orC₁₋₃ alkyl. In more suitable compounds of general formula (I), R⁴ is H,chloro, bromo, cyano or methyl. In particularly suitable compounds ofgeneral formula (I), R⁴ is H, chloro or methyl and especially H.

As set out above, R⁵ is H or methyl, more suitably H.

In some compounds of general formula (I), both R⁴ and R⁵ are H.

In other compounds of general formula (I), R⁴ is H and R⁵ is methyl.

In other compounds of general formula (I), R⁴ is methyl and R⁵ is H.

In other compounds of general formula (I), R⁴ is halo (e.g. chloro orbromo) and R⁵ is H.

In other compounds of general formula (I), R⁴ is cyano and R⁵ is H.

Some particularly suitable compounds of the present invention includethose having a cation selected from:

-   1.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-3-methyl-1H-1,3-benzodiazol-3-ium;-   2.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-6-fluoro-3-methyl-1H-1,3-benzodiazol-3-ium;-   3.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-chloro-1-ethyl-3-methyl-1H-1,3-benzodiazol-3-ium;-   4.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-6-methoxy-3-methyl-1H-1,3-benzodiazol-3-ium;-   5.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-3-methyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-ium;-   6.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-3-methyl-6-(trifluoromethoxy)-1H-1,3-benzodiazol-3-ium;-   7.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-methoxy-1H-1,3-benzodiazol-3-ium;-   8.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-3-benzyl-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-ium;-   9.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-6-methoxy-3-(2-methoxy-2-oxoethyl)-1H-1,3-benzodiazol-3-ium;-   10.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-3-(carboxylatomethyl)-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-ium-   11. 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)    methyl]-3-(carbamoylmethyl)-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-ium;-   12. 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)    methyl]-1-ethyl-6-methoxy-3-[2-(methylsulfanyl)ethyl]-1H-1,3-benzodiazol-3-ium;-   13. 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)    methyl]-1-ethyl-3-(2-hydroxyethyl)-6-methoxy-1H-1,3-benzodiazol-3-ium;-   14. 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)    methyl]-1-ethyl-3-{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}-6-methoxy-1H-1,3-benzodiazol-3-ium;-   15.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-benzyl-3-methyl-1H-1,3-benzodiazol-3-ium;-   16.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-3-benzyl-6-chloro-1-ethyl-1H-1,3-benzodiazol-3-ium;-   17.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-3-benzyl-1-ethyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-ium;-   18.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-3-benzyl-1-[2-oxo-2-(piperidin-1-yl)ethyl]-1H-1,3-benzodiazol-3-ium;-   19.    2-[({3-amino-6-methyl-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-6-methoxy-3-methyl-1H-1,3-benzodiazol-3-ium;-   20.    2-[({3-amino-7-methyl-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-chloro-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   21.    2-[({3-amino-7-chloro-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-chloro-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   22.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   23.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   24.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-{[(tert-butoxy)carbonyl]amino}propyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   25.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   26.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(1-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1H-pyrazol-4-yl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   27.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(2-aminoethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   28.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-aminopropoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   29.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-aminopropyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   30.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-(piperidin-4-yl)-1H-1,3-benzodiazol-3-ium;-   31.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]-1H-1,3-benzodiazol-3-ium;-   32.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(2-carbamimidamidoethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   33.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-carbamimidamidopropyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   34.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   35. 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)    methyl]-6-[(3-{[(tert-butoxy)carbonyl]amino}propyl)carbamoyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium    formic acid;-   36. 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)    methyl]-6-({1-[(tert-butoxy)carbonyl]piperidin-4-yl}carbamoyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   37. 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)    methyl]-6-(4-{[(tert-butoxy)carbonyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   38.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{[2-(4-{[(tert-butoxy)carbonyl]amino}piperidin-1-yl)ethyl]carbamoyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   39.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3-aminopropyl)carbamoyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   40.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-[(piperidin-4-yl)carbamoyl]-1H-1,3-benzodiazol-3-ium;-   41.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-aminopiperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   42.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{[2-(4-aminopiperidin-1-yl)ethyl]carbamoyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   43.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-5-(2-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}ethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   44.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-5-(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   45.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-5-(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   46.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-{1-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]piperidin-4-yl}-1H-1,3-benzodiazol-3-ium;-   47.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-(1-{1-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]piperidin-4-yl}-1H-pyrazol-4-yl)-1H-1,3-benzodiazol-3-ium;-   48.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)carbamoyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   49.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-({1-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]piperidin-4-yl}carbamoyl)-1H-1,3-benzodiazol-3-ium;-   50. 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)    methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   51. 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)    methyl]-6-{[2-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidin-1-yl)ethyl]carbamoyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   52.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-5-[4-({bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}methyl)piperidine-1-carbonyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   53.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3R)-3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}pyrrolidine-1-carbonyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   54.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3S)-3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}pyrrolidine-1-carbonyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   55.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-{[(1r,4r)-4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}cyclohexyl]carbamoyl}-1H-1,3-benzodiazol-3-ium;-   56.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-{[(1s,4s)-4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}cyclohexyl]carbamoyl}-1H-1,3-benzodiazol-3-ium;-   57.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)(methyl)carbamoyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   58.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(2-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}ethyl)carbamoyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   59.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-5-{[(14S,15R,16R,17R)-14,15,16,17,18-pentahydroxy-12-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]-3,6,9-trioxa-12-azaoctadecan-1-yl]carbamoyl}-1H-1,3-benzodiazol-3-ium;-   60.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-({2-[4′-(2-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}ethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamoyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   61.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{[(3S)-3-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)amino]-3-carbamoylpropyl]carbamoyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   62.    2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{[4-(4-{3-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)amino]-3-carbamoylpropyl}phenyl)butyl]carbamoyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   63.    2-[({3-amino-7-chloro-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   64.    2-[({3-amino-7-bromo-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   65.    2-[({3-amino-7-chloro-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   66.    2-[({3-amino-7-bromo-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;-   67.    2-[({3-amino-7-cyano-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-methoxy-1H-1,3-benzodiazol-3-ium;-   and an anion X⁻ as defined for general formula (I).

Compounds of general formula (I) may be prepared by reacting a compoundof general formula (II) or a salt or activated derivative thereof:

wherein R⁴ and R⁵ are as defined for general formula (I);with a salt of general formula (III):

wherein R¹, R² and R³ are as defined for general formula (I);and X⁻ is as defined for general formula (I) and which may be the sameor different from the X⁻ of the product of general formula (I).

Suitably the reaction is carried out under basic conditions in thepresence of a coupling reagent, which may generate an activated acid asan intermediate. The basic conditions may be supplied by anon-nucleophilic base such as N,N-diisopropylethylamine (DIPEA) ortrimethylamine. Suitable coupling reagents includeO-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU),O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU), N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate (HBTU) or a combination of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) with1-hydroxy-7-azabenzotriazole (HOAt) or hydroxybenzotriazole (HOBt).

In some cases, carbonyldiimidazole (CDI) may be used as a couplingagent.

In other cases, imidazole or a salt thereof (e.g. imidazolehydrochloride) may be used.

The reaction may be conducted at a temperature of about 10 to 50° C.,more usually at 15 to 30° C., or room temperature and in an organicsolvent such as N,N-dimethylformamide.

When a CDI coupling agent is used, a compound of general formula (I) maybe prepared by reacting a compound of general formula (III) as definedabove with an activated compound of general formula (IV):

wherein R⁴ and R⁵ are as defined for general formula (I).

As discussed above, the reaction is suitably conducted at a temperatureof about 10 to 50° C., more usually at 15 to 30° C., or room temperatureand in an organic solvent such as N,N-dimethylformamide.

The activated compounds of general formula (IV) are prepared by reactinga compound of general formula (II) as defined above or a salt thereofwith carbonyl diimidazole (CDI). Suitably the reaction takes place in anorganic solvent such as N,N-dimethylformamide and at a temperature offrom about 10 to 30° C., more usually 15 to 25° C. or room temperature.

In cases where the target compound of general formula (I) has an R¹, R²,R³, R⁴ and/or R⁵ substituent which is sensitive to the conditions usedwhen the compound of general formula (III) is reacted with a compound ofgeneral formula (II) or an activated derivative thereof, for example acompound of general formula (IV), the compounds of general formulae(III) may comprise a protected R¹, R² and/or R³ substituent and/or thecompounds of general formulae (II) and (IV) may comprise a protected R⁴and/or R⁵ substituent. This is illustrated in Example 46, where acompound of general formula (IV) is reacted with a protected derivativeof a compound of general formula (III) in which R¹ is a group LR¹⁰,where L is a group -Q¹-Z¹—, where Z¹ is —CH₂[CH(OH)]₅— and R¹⁰ is H(Intermediate 98) to give a protected derivative of general formula (I)(Intermediate 99). In Intermediates 98 and 99, two of the OH groups areprotected by forming a substituted 1,3-dioxanyl group. This protectinggroup may be removed by treatment with aqueous acid, e.g. aqueoushydrochloric acid (see Example 46). Similarly, Intermediates 89 and 93,which are compounds of general formula (III) are prepared bydeprotecting Intermediates 88 and 92 respectively.

Compounds of general formula (II) may be prepared by hydrolysis of acompound of general formula (V):

wherein R⁴ and R⁵ are as defined for general formula (I) and R²¹ is C₁₋₆alkyl or benzyl.

Suitably, the hydrolysis is base hydrolysis such that the compound ofgeneral formula (V) is reacted with a base, suitably a strong aqueousbase such as lithium hydroxide, potassium hydroxide or sodium hydroxide.

Compounds of general formula (V) wherein R⁴ and R⁵ are H and R²¹ is C₁₋₆alkyl or benzyl. may be prepared by cyclising a compound of generalformula (XX):

wherein each R²¹ is independently C₁₋₆ alkyl or benzyl.

Suitably, the cyclisation is acid mediated, suitably the acid is aceticacid at elevated temperature such as 80° C.

Compounds of general formula (XX) may be prepared by palladium mediatedcoupling of a vinyl boronate ester with a compound of formula (XXI):

wherein R²¹ is as defined above and R²² is halo, particularly Cl, Br orI.

Compounds of general formula (XXI) are well known and are commerciallyavailable or may be prepared by methods familiar to those of skill inthe art.

Some compounds of general formula (V) may be converted to othercompounds of general formula (V). For example, compounds of generalformula (V) in which R⁴ is halo may be prepared from compounds ofgeneral formula (V) in which R⁴ is H by reaction with a halogenatingagent such as N-chlorosuccinimide, N-bromosuccinimide orN-iodosuccinimide. Suitable reaction conditions are described in thepreparation of Intermediates 10, 14 and 161. In some cases, for exampleas shown in the synthesis of Intermediate 161, the nitrogen at the5-position of the pyrrolopyrazine ring may be protected with atrimethylsilylethoxymethyl protecting group.

Compounds of general formula (V) in which R⁴ is halo may be convertedinto compounds of general formula (V) in which R⁴ is alkyl or cyano.Compounds in which R⁴ is alkyl may be prepared from compounds in whichR⁴ is halo by reaction with an organometallic reagent, for exampledialkyl zinc, as described for the preparation of Intermediate 11 below.

Compounds of general formula (V) in which R⁴ is cyano may be preparedfrom compounds of general formula (V) in which R⁴ is halo by reactionwith potassium ferrocyanide, as described for the preparation ofIntermediate 166. For this reaction, the nitrogen at the 5-position ofthe pyrrolopyrazine ring should be protected, for example with atrimethylsilylethoxymethyl protecting group.

Compounds of general formula (V) in which R⁴ is C₁₋₆ alkyl and R⁵ is Hmay be prepared by transition metal mediated coupling of the appropriatedialkyl zinc to a compound of general formula (V) in which R⁴ is halo.Suitably, the transition metal is a Palladium (0) complex.

Compounds of general formula (V) in which R⁴ is H and R⁵ is methyl maybe prepared by transition metal mediated cyclisation of a compound ofgeneral formula (XXII).

wherein R²¹ is as defined above.

Suitably, the transition metal is a Palladium (0) complex. Suitably thecyclisation is carried out in the presence of copper (I) iodide and abase.

Compounds of general formula (XXII) may be prepared by transition metalmediated coupling of a compound of general formula (XXI) with apropargyl silane.

Compounds of general formula (III) may be prepared from compounds ofgeneral formula (VII)

-   -   wherein R¹, R² and R³ are as defined for general formula (I) and        X⁻ is as defined for general formula (I) and which may be the        same or different from the X⁻ of the product of general formula        (III);    -   R²³ is O(C₁₋₆) alkyl optionally substituted with aryl; or    -   aryl optionally substituted with C(O)OH; and    -   R²⁴ is H; or    -   R²³ and R²⁴ together with the carbon and nitrogen atoms to which        they are attached form a heterocyclic ring, optionally fused to        an aryl or heteroaryl ring and optionally containing a further        ═O substituent.

In some cases, the removal of the protecting group R²³ may be achievedby reaction with an acid. This is appropriate for alkyloxycarbonylprotecting groups, for example when R²³ is ^(t)butyloxy. Reaction withan acid may result in a change in the anion X⁻. Furthermore, followingreaction with an acid, the compound of formula (III) will usually bepresent in the form of its acid addition salt.

Other protecting groups, for example Fmoc (i.e. when R²³ isfluorenylmethyloxy), can be removed by treatment with a base, forexample piperidine or morpholine.

In some suitable compounds of general formula (VII), R²³ is benzyloxy orfluoren-9-ylmethyloxy and R¹ comprises another protecting group, forexample ^(t)butyloxycarbonyl (Boc), such that the two protecting groupsare stable under different conditions.

Examples of cyclic N(R²⁴)C(O)R²³ groups include1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl. Examples of individual R²³groups include methoxy, ethoxy, ^(n)propoxy, ^(i)propoxy, ^(n)butyloxy,^(s)butyloxy, ^(t)butyloxy, benzyloxy, fluorenylmethyloxy and phenyloptionally substituted with C(O)OH.

When R²³ and R²⁴ together with the carbon and nitrogen atoms to whichthey are attached form a heterocyclic ring of this type, the compound ofgeneral formula (IIIA) may be synthesised by reacting the compound ofgeneral formula (VII) with hydrazine hydrate. Suitably, this reaction iscarried out in an alcoholic solvent such as methanol and at elevatedtemperature, for example about 60-90° C., typically about 75° C.

Some compounds of general formula (VII) are known. For example,Intermediate 58 may be synthesised according to the procedure describedin US 2015/0018313)

Other compounds of general formula (VII) may be prepared from compoundsof general formula (VIII):

wherein R¹ and R³ are as defined for general formula (I) and R²³ and R²⁴are as defined for general formula (VII);by reaction with a compound of general formula (IX):R²—X¹  (IX)wherein R² is as defined for general formula (I) and X¹ is a leavinggroup such as halo; or with a compound of general formula (IXA):R^(2a)—X¹  (IXA)wherein X¹ is as defined above for general formula (IX) and R^(2a) is aprotected R² group. For example, when the desired R² group contains oneor more OH moieties, these may be protected using standard protectinggroups, for example silyl protecting groups such as trimethylsilyl(TMS), ^(t)butyldimethylsilyl (TBDMS) etc.

When the route via the compound of general formula (IXA) is used, thecompound of general formula (VIII) is suitably one in which R²³ is—O(C₁₋₆) alkyl optionally substituted with aryl, or R²³ is aryloptionally substituted with C(O)OH; and R²⁴ is H; because, in this case,the silyl and carbonyloxy protecting groups can be removed using an acidsuch as hydrogen chloride solution.

In some cases, when a compound of general formula (IX) is reacted with acompound of general formula (VIII) in which N(R²⁴)C(O)R²³ is a cyclicgroup such as 1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl, the reaction maybe accompanied by ring opening. Thus a compound of general formula(VIII) in which N(R²⁴)C(O)R²³ is 1,3-dioxo-2,3-dihydro-1H-isoindol-2-ylmay give rise to a product of general formula (VII) in whichN(R²⁴)C(O)R²³ is:

Some compounds of general formula (VIII) are known. For example,Intermediate 23 (see examples below) may be synthesised by the route setout in US 2015/0018314 A1.

Some compounds of general formulae (VII) and (VIII) may be synthesisedfrom other compounds of general formulae (VII) and (VIII). For example,a compound of general formula (VIII) in which R¹ is C(O)OR⁵, where R⁵ isother than H may be converted to a compound of general formula (VIII) inwhich R¹ is C(O)OH by hydrolysis, for example with a base, suitably analkali metal hydroxide such as lithium hydroxide. The compound in whichR¹ is C(O)OH may then be converted to a compound in which R¹ isC(O)OR¹², where R¹² is other than H by reaction with a compound ofgeneral formula (X):

wherein R¹² is as defined for general formula (I) except that it is notH and R²⁵ is C₁₋₄ alkyl.

This type of conversion is exemplified below in the synthesis ofIntermediate 81.

Compounds of general formula (VIII) can also be converted to othercompounds of general formula (VIII) with a different R²³ and/or R²⁴groups. For example, when R²³ is O(C₁₋₆) alkyl optionally substitutedwith aryl, or R²³ is aryl optionally substituted with C(O)OH; and R²⁴ isH, the compound of general formula (VIII) may be hydrolysed, for exampleby reaction with HCl in a solvent such as dioxane, to give a compound ofgeneral formula (XI):

wherein R¹ and R³ are as defined in general formula (I). This isexemplified by the synthesis of Intermediate 79 from Intermediate 78.

The compound of general formula (XI) may be re-protected to obtain a newcompound of general formula (VIII), for example by reaction with acompound of general formula (XII):

wherein R²³ is as defined above for general formula (VII). The reactionmay be conducted in the presence of a base such as trimethylamine in apolar organic solvent, for example dichloromethane and at a temperatureof from 10 to 30° C., more usually 15 to 25° C., typically at roomtemperature. An example of this type of process is the synthesis ofIntermediate 80 from Intermediate 79

Compounds of general formula (VII) and (VIII) may also be synthesisedfrom other compounds of general formula (VII) and (VIII) using themethods described for the preparation of Intermediates 66, 67, 70, 71,74, 81, 86, 87, 90, 91, 96, 97, 157 and 159 below.

Thus, for example a compound of general formula (VII) or (VIII) in whichR¹ is halo, particularly bromo, may be reacted with an alkyne to give acompound of general formula (VII) in which R¹ is -L¹R¹⁰, where L¹comprises an alkynylene group. The reaction may be catalysed with acopper (I) salt, for example copper (I) iodide. This is illustrated inthe synthesis of Intermediate 66.

A compound of general formula (VII) or (VIII) in which R¹ is halo,particularly bromo, may also be reacted with a compound of generalformula (XX):R^(1a)—X²  (XX)where R^(1a) is as defined above for R¹ except that it is not halo andX² is an organoborane group, for example4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl. A reaction of this type isused in the preparation of Intermediates 70 and 74.

Compounds of general formulae (VII) and (VIII) in which R¹ is -L¹R¹⁰,where L¹ comprises an alkenylene, alkynylene or partially saturatedcarbocyclyl or heterocyclyl moiety may be reduced, suitably by catalytichydrogenation, to give compounds of formula (VII) in which L¹ comprisesan alkylene or saturated carbocyclyl or heterocyclyl moiety. Examples ofthis are shown in the synthesis of Intermediates 67 and 71 below.

Compounds of general formulae (VII) and (VIII) in which R¹ is C(O)OH maybe esterified to give compounds of general formulae (VII) and (VIII) inwhich R¹ is C(O)OR¹², where R¹² is as defined above except that it isnot H. An example of this is the preparation of Intermediate 81, inwhich the carboxylic acid derivative Intermediate 80 is reacted with1,1-di-tert-butoxy-N,N-dimethylmethanamine.

Compounds of general formulae (VII) and (VIII) in which R¹ or R³ (forcompounds of general formula (VIII)) comprises a —C(O)OR¹² or —C(O)OR⁷group in which R¹² or R⁷ is other than H; or a —C(O)N(R⁷)R⁸ group can beconverted to compounds in which R¹ or R³ comprises a —C(O)OH group byhydrolysis. In some cases, base hydrolysis may be used, for exampleusing a base such as lithium hydroxide, sodium hydroxide or potassiumhydroxide. Alternatively, the hydrolysis may be acid hydrolysis using anacid such as hydrochloric acid. This is particularly suitable when theR¹² or R⁷ group is an alkyl group such as tert-butyl.

Compounds of general formulae (VII) and (VIII) in which R¹ is C(O)OH maybe converted to compounds in which R¹ is —C(O)NR¹²R¹³, wherein R¹² andR¹³ are as defined above, or in which R¹ is -L¹R¹⁰, wherein

-   -   L¹ is    -   —C(O)N(R⁷)Z¹—, —C(O)N(R⁷)Q¹-, —C(O)N(R⁷)Z¹Q¹-,        —C(O)N(R⁷)Z¹Q¹Z²—, —C(O)N(R⁷)Q¹Z¹—, —C(O)N(R⁷)Q¹Q²-,        —C(O)N(R⁷)Q¹Q²Z¹—, —C(O)N(R⁷)Z¹Q¹Q²Z²—,        —C(O)N(R⁷)Z¹O(CH₂CH₂O)_(n)Z²—, —C(O)N(R⁷)Z¹O(CH₂O)_(n)Z²—,        —C(O)N(R⁷)Z¹Q¹Z²N(R⁸)Z³—, —C(O)N(R⁷)Z¹N(R⁸)Z²—,        —C(O)N(R⁷)Q¹Z¹N(R⁸)Z²—, —C(O)N(R⁷)Z¹Q¹OQ²OQ³- or        —C(O)N(R⁷)Z¹Q¹OQ²OQ³Z²—;    -   especially    -   —C(O)N(R⁷)Z¹—, —C(O)N(R⁷)Q¹-, —C(O)N(R⁷)Z¹Q¹-,        —C(O)N(R⁷)Z¹Q¹Z²—, —C(O)N(R⁷)Q¹Z¹—, —C(O)N(R⁷)Q¹Q²- or        —C(O)N(R⁷)Q¹Q²Z¹—;    -   by reaction with an appropriate amine or ammonium salt.

Compounds of general formulae (VII) and (VIII) in which R¹ is L¹R¹⁰where R¹⁰ is —N(R⁷)—C(O)OR⁸ can be converted to compounds of generalformulae (VII) and (VIII) in which R¹ is L¹R¹⁰ where R¹⁰ is —NH₂ byhydrolysis, for example acid hydrolysis using hydrochloric acid in asolvent such as dioxane. An example of this is the synthesis ofIntermediate 86.

These compounds can in turn be converted to compounds of generalformulae (VII) and (VIII) in which R¹⁰ is N(H)R⁷ or N(R⁷)R⁸ where R⁷ isCH₂—R^(7a) and R⁸ is CH₂—R^(8a) and each R^(7a) and R^(8a) isindependently selected from H and C₁₋₁₁ alkyl optionally substitutedwith one or more halo or OH groups or protected OH groups. Theconversion can be achieved by reductive amination using a reducing agentsuch as a hydride, for example sodium cyanoborohydride, with an aldehydeor acetal as shown below:

-   -   where L¹ is as defined for general formula (I);    -   R⁷ is —CH₂R^(7a), where R^(7a) is H or C₁₋₁₁ alkyl optionally        substituted with one or more halo or OH groups; and    -   R¹⁹ is C₁₋₁₂ alkyl; and

A is a fragment of a compound of general formula (VII) or (VIII) notincluding R¹ as follows:

In other cases, a cyclic hemiacetal can be used in place of the aldehydeor acetal. The scheme below shows an example where a 6-memberedhemiacetal is used to give a compound in which R¹ is L¹-NHR⁷ where R⁷ is(CH₂)₄CH₂OH:

wherein A and L¹ are as defined above.

If a large excess of the aldehyde, acetal or cyclic hemiacetal is used,both of the amine hydrogen atoms will be replaced. In some cases, it maybe possible to react successively with different aldehydes, acetals orhemicacetals to yield compounds in which R⁷ and R⁸ are different.

More suitably in these product compounds of general formulae (VII) or(VIII) both R^(7a) and R^(8a) are C₁₋₁₁ alkyl optionally substitutedwith one or more halo or OH or protected OH groups; and most suitablyeach R⁷ and R⁸ is CH₂[CH(OH)]₄CH₂OH, wherein OH groups are optionallyprotected, for example as acetals, such as benzylidene acetals.

Examples of this type of reaction include the conversion of Intermediate86 to Intermediate 87 and the conversion of Intermediate 90 toIntermediate 91.

Similarly, compounds of general formulae (VII) and (VIII) in which R¹ isL¹R¹⁰ where: Similarly, compounds of general formulae (VII) and (VIII)in which L¹ comprises a moiety Q¹, Q² or Q³ linked to R¹⁰ via a ringnitrogen atom and in which R¹⁰ is C(O)OR⁷; may be converted to compoundsin which R¹⁰ is H by hydrolysis, for example acid hydrolysis usinghydrochloric acid in a solvent such as dioxane.

An example of a reaction of this type is the preparation of Intermediate96 from Intermediate 95.

Compounds of general formulae (VII) and (VIII) in which L¹ comprises amoiety Q¹, Q² or Q³ linked to R¹⁰ via a ring nitrogen atom and in whichR¹⁰ is H can be converted to compounds of general formula (I) in whichthe Q¹, Q² or Q³ moiety is linked is linked to a Z¹, Z² or Z³ moiety,wherein Z¹, Z² or Z³ is:

CH₂—C₁₋₁₁ alkylene, CH₂—C₂₋₁₁ alkenylene, CH₂—C₂₋₁₁ alkynylene any ofwhich is optionally substituted by one or more halo, OH, C(O)NR¹⁵R¹⁶,C(O)OR¹⁵ or NR¹⁵R¹⁶; and each R¹⁵ and R¹⁶ is independently H or C₁₋₆alkyl;

-   -   and R¹⁰ is H;    -   by reductive amination with an aldehyde, acetal or cyclic        hemiacetal equivalent compound using a method similar to that        described above for the conversion of compounds of general        formulae (VII) and (VIII) in which R¹ is L¹NH₂ to compounds of        general formulae (VII) and (VIII) in which R¹ is L¹N(R⁷)R⁸ where        R⁷ is CH₂—R^(7a) and R⁸ is CH₂—R^(8a) and each R^(7a) and R^(8a)        is independently selected from H and C₁₋₁₁ alkyl optionally        substituted with one or more halo or OH groups.

More suitably in these product compounds of general formulae (VII) or(VIII) Z¹, Z² or Z³ is CH₂—C₁₋₁₁ alkyl optionally substituted with oneor more halo or OH groups; and most suitably is CH₂[CH(OH)]₄CH₂OH,wherein OH groups are optionally protected, for example as acetals, suchas benzylidene acetals.

An example of this process is shown in the preparation of Intermediate97.

Compounds of general formulae (VII) and (VIII) in which R¹ is L¹R¹⁰ andR¹⁰ is NH₂ can be converted into compounds in which R¹⁰ is—NHC(═NR⁹)N(R⁸)₂ by reaction with a carboximidamide or a salt thereof,for example 1,2,4-triazole carboximidamide hydrochloride.

Compounds of general formula (VIII) may also be prepared from compoundsof general formula (XIII):

wherein R¹ and R³ are as defined for general formula (I);by reaction with a compound of general formula (XIV):

wherein R²³ is as defined above for general formula (VII).

The reaction suitably takes place in the presence of a base, typically anon-nucleophilic base, for example an amine such asN,N-diisopropylethylamine (DIPEA) or triethylamine and a peptidecoupling agent, for example HATU, TBTU, HBTU or a combination of EDCwith HOAt or HOBt. The reaction is suitably conducted at a temperatureof about 10 to 30° C., usually 15 to 25° C., for example at roomtemperature. Suitable reaction solvents include organic solvents such asN,N-dimethylformamide (DMF).

Compounds of general formulae (XIII) and (XIV) are known and are readilyavailable or may be prepared by methods known to those of skill in theart.

Alternatively, compounds of general formula (XIII) may be prepared fromcompounds of general formula (XV):

wherein R¹ and R³ are as defined for general formula (I);by catalytic hydrogenation, suitably using a palladium catalyst.

The hydrogenation is suitably carried out at 1 atmosphere pressure andat a temperature of about 10 to 30° C., usually 15 to 25° C., forexample at room temperature.

The product of general formula (XIII) can be reacted directly with acompound of general formula (XIV) as described above without furtherisolation or purification steps.

Compounds of general formula (XV) may be prepared from compounds ofgeneral formula (XVI):

wherein R¹ is as defined for general formula (I) and X² is a leavinggroup, particularly a halo group such as chloro or fluoro;by reaction with a compound of general formula (XVII):R³—NH₂  (XVII)wherein R³ is as defined for general formula (I).

The reaction is suitably carried out under pressure, at a temperature ofabout 30-70° C., more usually about 40-60° C., typically about 50° C.and in an organic solvent such as tetrahydrofuran.

Compounds of general formulae (XVI) and (XVII) are known and are readilyavailable or may be prepared by methods known to those of skill in theart.

An alternative method for the preparation of a compound of generalformula (I) is by reaction of a compound of general formula (XVIII):

wherein R¹, R³, R⁴ and R⁵ are as defined for general formula (I);with a compound of general formula (IX) or (IXA) as defined above underconditions similar to those described above for the reaction of thecompound of general formula (VIII) with the compound of general formula(IX) or (IXA).

Compounds of general formula (I) may also be synthesised from othercompounds of general formula (I), for example using the methodsdescribed in Examples 10, 27-33, 35-42 and 47-62 below. In general,methods of converting compounds of general formulae (VI) and (VIII) toother compounds of general formulae (VI) and (VIII) may also be appliedto compound of general formula (I) and vice versa.

Thus, for example, compounds of general formula (I) in which R³comprises a —C(O)OR⁷ group in which R⁷ is other than H or a —C(O)N(R⁷)R⁸group can be converted to compounds in which R³ comprises a —C(O)OH orC(O)O— group by hydrolysis. In some cases the hydrolysis may be basehydrolysis, for example using a base such as lithium hydroxide, sodiumhydroxide or potassium hydroxide as shown in Example 10. Alternatively,acid hydrolysis may be employed, for example using hydrochloric acid.

Compounds of general formula (I) in which R¹ is L¹R¹⁰ where R¹⁰ is—N(R⁷)—C(O)OR⁸ can be converted to compounds of general formula (I) inwhich R¹ is L¹R¹⁰ where R¹⁰ is —NHR⁷ by hydrolysis, for example acidhydrolysis using hydrochloric acid in a solvent such as dioxane.Suitably R⁷ is H such that in the product compound of general formula(I) R¹⁰ is NH₂. This is illustrated in Examples 27-29, 39, 41 and 42.

Compounds of general formula (I) in which R¹ is LR¹⁰, wherein L¹comprises a moiety Q¹, Q² or Q³ which is linked to R¹⁰ via a ringnitrogen atom and in which R¹⁰ is C(O)OR⁷ can be converted to compoundsof formula (I) in which R¹⁰ is H by a similar method, i.e. byhydrolysis, for example acid hydrolysis using hydrochloric acid in asolvent such as dioxane. This is illustrated in Examples 30, 31 and 40.

The compounds of formula (I) in which R¹ is LR¹⁰, and R¹⁰ is NH₂ can inturn be converted to compounds of general formula (I) in which R¹⁰ isN(R⁷)R⁸ where R⁷ is CH₂—R^(7a) and R⁸ is CH₂—R^(8a) and one of R^(7a)and R^(8a) is C₁₋₁₁ alkyl optionally substituted with one or more haloor OH groups; and the other of R^(7a) and R^(8a) is H or C₁₋₁₁ alkyloptionally substituted with one or more halo or OH groups. Similarly,the compounds of general formula (I) in which R¹ is LR¹⁰, and L¹comprises a moiety Q¹, Q² or Q³ linked to R¹⁰ via a ring nitrogen atomand in which R¹⁰ is H can be converted to compounds of general formula(I) in which Q¹, Q² or Q³ is linked to a Z¹, Z² or Z³ moiety via a ringnitrogen atom, wherein Z¹, Z² or Z³ is CH₂—C₁₋₁₁ alkyl optionallysubstituted with one or more halo or OH groups; and R¹⁰ is H. Theseconversions can be achieved by reductive amination with an aldehyde,acetal or cyclic hemiacetal equivalent compound using a similar methodto that described above for the compounds of general formulae (VII) and(VIII). Examples of this type of reaction include the methods of:

Example 45, where the compound of Example 29, in which L¹ is a moiety—Z¹— and R¹⁰ is NH₂ is reacted with 4,6-O-benzylidene-D-glucopyranose togive a product where R¹⁰ is —N{CH₂[CH(OH)]₄—CH₂OH}₂; and

Example 47, where the compound of Example 31, where L¹ is a moiety-Q¹Q²- in which Q² is linked to R¹⁰ via a ring nitrogen atom and R¹⁰ isH is reacted with 4,6-O-benzylidene-D-glucopyranose to give a product inwhich L¹ is Q¹Q²Z¹, where Z¹ is CH₂[CH(OH)]₄CH₂O—; and R¹⁰ is H.

Compounds in which R¹ is L¹R¹⁰ and R¹⁰ is NH₂ can be converted intocompounds in which R¹⁰ is —NHC(═NR⁹)N(R⁸)₂ by reaction with acarboximidamide or a salt thereof, for example 1,2,4-triazolecarboximidamide hydrochloride. This process is shown in Examples 32 and33.

Compounds in which R¹ is C(O)OH may be converted to compounds in whichR¹ is —C(O)NR¹²R¹³, wherein R¹² and R¹³ are as defined above, or inwhich R¹ is -L¹R¹⁰, wherein

-   -   L¹ is —C(O)N(R⁷)Z¹—, —C(O)N(R⁷)Q¹-, —C(O)N(R⁷)Z¹Q¹-,        —C(O)N(R⁷)Z¹Q¹Z²—, —C(O)N(R⁷)Q¹Z¹—, —C(O)N(R⁷)Q¹Q²-,        —C(O)N(R⁷)Q¹Q²Z¹—, —C(O)N(R⁷)Z¹Q¹Q²Z²—,        —C(O)N(R⁷)Z¹O(CH₂CH₂O)_(n)Z²— —C(O)N(R⁷)Z¹O(CH₂O)_(n)Z²—,        —C(O)N(R⁷)Z¹Q¹Z²N(R⁸)Z³—, —C(O)N(R⁷)Z¹N(R⁸)Z²—,        —C(O)N(R⁷)Q¹Z¹N(R⁸)Z²—, —C(O)N(R⁷)Z¹Q¹OQ²OQ³-,        —C(O)N(R⁷)Z¹Q¹OQ²OQ³Z²—; or    -   L¹ is —C(O)Q¹-, —C(O)Q¹Z¹—, —C(O)Q¹Q²-, —C(O)Q¹Q²Z¹—,        —C(O)Q¹N(R⁷)C(O)Z¹—, —C(O)Q¹N(R⁷)C(O)Z¹Q²-,        —C(O)Q¹N(R⁷)C(O)Z¹Q²Q³- or —C(O)Q¹NR(R⁷)C(O)Z¹Q²Z²—, wherein Q¹        is a heterocyclyl ring linked to the —C(O) moiety via a ring        nitrogen atom;        by reaction with an appropriate amine or ammonium salt.

Suitably the reaction is carried out under basic conditions in thepresence of a coupling reagent. The basic conditions may be supplied bya non-nucleophilic base such as N,N-diisopropylethylamine (DIPEA) ortrimethylamine. Suitable coupling reagents includeO-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU),O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU), N,N,N′,N′ tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate (HBTU) or a combination of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) with1-hydroxy-7-azabenzotriazole (HOAt) or hydroxybenzotriazole (HOBt).

This type of reaction is illustrated in Examples 35 to 38, 48-60, 65 and66.

In some compounds of the invention, such as Example 35, L¹ is—C(O)N(R⁷)Z¹— where R⁷ is H and Z¹ is as defined above; R¹⁰ is—N(R⁷)—C(O)OR⁸; R⁷ is H and R⁸ is as defined above.

In some compounds of the invention, such as Example 36, L¹ is—C(O)N(R⁷)Q¹- where R⁷ is H and Q¹ is heterocyclyl linked to R¹⁰ via aring nitrogen atom; R¹⁰ is —C(O)OR⁷ where R⁷ is as defined above.

In some compounds of the invention, such as Example 37, L¹ is —C(O)Q¹-where Q¹ is heterocyclyl linked to C(O) via a ring nitrogen atom and toR¹⁰ via a ring carbon atom; R¹⁰ is —N(R⁷)—C(O)OR⁸; R⁷ is H and R⁸ is asdefined above.

In some compounds of the invention, such as Example 38, L¹ is—C(O)N(R⁷)Z¹Q¹- where R⁷ is H, Z¹ is as defined above, Q¹ isheterocyclyl linked to R¹⁰ via a ring carbon atom; R¹⁰ is—N(R⁷)—C(O)OR⁸; R⁷ is H and R⁸ is as defined above.

In some compounds of the invention, such as Examples 48, 57 and 58, L¹is C(O)N(R⁷)Z¹ where R⁷ is H or methyl and Z¹ is as defined above; R¹⁰is N(R⁷)R⁸, where R⁷ and R⁸ are both C₁₋₁₂ alkyl substituted with one ormore OH groups.

In some compounds of the invention, such as Example 49, L¹ isC(O)N(R⁷)Q¹Z¹ where R⁷ is H, Q¹ is linked to —C(O)N(R⁷)— via a ringcarbon atom and to Z¹ via a ring nitrogen, Z¹ is C₁₋₁₂ alkylenesubstituted with one or more OH; R¹⁰ is H.

In some compounds of the invention, such as Examples 50, 53, 54, 65 and66, L¹ is C(O)Q¹, where Q¹ is linked to C(O) via a ring nitrogen atom;R¹⁰ is N(R⁷)R⁸, where R¹ and R⁸ are both C₁₋₁₂ alkyl substituted withone or more OH groups.

In some compounds of the invention, such as Example 51, L¹ is—C(O)N(R⁷)Z¹Q¹- where R⁷ is H; Z¹ is as defined above, Q¹ is linked toR¹⁰ via a ring carbon atom, R¹⁰ is N(R⁷)R⁸, where R⁷ and R⁸ are bothC₁₋₁₂ alkyl substituted with one or more OH groups.

In some compounds of the invention, such as Example 52, L¹ is —C(O)Q¹Z¹—where Q¹ is linked to the C(O) moiety via a ring nitrogen atom and Z¹ asdefined above; R¹⁰ is N(R⁷)R⁸, where R⁷ and R⁸ are both C₁₋₁₂ alkylsubstituted with one or more OH groups.

In some compounds of the invention, such as Examples 55 and 56, L¹ is—C(O)N(R⁷)Q¹- where R⁷ is H, Q¹ is linked to —C(O)N(R⁷)— via a ringcarbon atom and to R¹⁰ via a ring nitrogen atom; R¹⁰ is N(R⁷)R⁸, whereR⁷ and R⁸ are both C₁₋₁₂ alkyl substituted with one or more OH groups.

In some compounds of the invention, such as Example 59, L¹ is—C(O)N(R⁷)Z¹O(CH₂CH₂O)_(n)Z², where R⁷ is H, Z¹, n and Z² are as definedabove; R¹⁰ is N(R⁷)R⁸, where R⁷ and R⁸ are both C₁₋₁₂ alkyl substitutedwith one or more OH groups.

In some compounds of the invention, such as Example 60, L¹ is—C(O)N(R⁷)Z¹Q¹Q²Z²—, where R⁷ is H, Z¹, Q¹, Q² and Z² are as definedabove; R¹⁰ is N(R⁷)R⁸, where R⁷ and R⁸ are both C₁₋₁₂ alkyl substitutedwith one or more OH groups.

The amines or ammonium salts which react with the compounds of generalformulae (I), (VII) or (VIII) in which R¹ is C(O)OH are described below.For example, an amine of general formula (XXX):

wherein R⁷ and R⁸ are as defined for general formula (I);L¹ is as defined for general formula (I) and is linked to H via an amineof a Z¹ moiety or a ring nitrogen atom of a Q¹ moiety;may be prepared from a protected compound of general formula (XXXI):

wherein R⁷ and R⁸ are as defined for general formula (I), L¹ is asdefined for general formula (XXX) and R³⁰ is an amine protecting groupsuch as fluorenylmethyloxycarbonyl (Fmoc), butyloxycarbonyl (Boc) orbenzyloxycarbonyl (Cbz). Deprotection may be by hydrogenation forcompounds where R³⁰ is Cbz or by reaction with an acid such ashydrochloric or hydrobromic acid in the case where R³⁰ is a protectinggroup such as Boc or Cbz or with a weak base such as morpholine orpiperidine when R³⁰ is a protecting group such as Fmoc.

When R⁷ and/or R⁸ is a group —CH₂[CH(OH)]₄CH₂OH, this may be protectedin the compound of general formula (XXXI), for example as a benzylideneacetal.

An amine of general formula (XXXI) in which R⁷ and/or R⁸ is a group—CH₂[CH(OH)]₄CH₂OH, protected as a benzylidene acetal may be reactedwith a compound of general formula (VII) or (VIII). In the resultingproduct, when the protecting group R²³ is an acid labile group such asBoc, the protecting group R²³ and the benzylidene acetal protectinggroups can be removed simultaneously using an acid. In some cases,however, a two step deprotection process may be used wherein the R²³group is removed by hydrogenation and the benzylidene acetal issubsequently removed by treatment with an acid. When the protectinggroup R²³ is a group such as Fmoc it may be removed by treatment with abase as described above. Treatment with an acid will then be required toremove the benzylidene acetal protection.

The preparation of other intermediates which can be used to react withcompounds of general formulae (I), (VII) or (VIII) to convert them toother compounds of general formulae (I), (VII) or (VIII) is describedbelow.

The compounds of general formula (I) are ENaC blockers and are thereforeuseful in the treatment or prevention of respiratory diseases andconditions.

Therefore in a further aspect of the invention there is provided acompound of general formula (I) for use in medicine.

Suitably, the compound of general formula (I) is for use in thetreatment or prophylaxis of a disease or condition mediated by ENaC.

There is also provided:

-   -   A compound of general formula (I) for use in the treatment or        prophylaxis of respiratory diseases and conditions.    -   A compound of general formula (I) for use in the treatment or        prophylaxis of skin conditions or ocular conditions.

The invention further provides:

-   -   The use of a compound of general formula (I) in the preparation        of a medicament for the treatment or prophylaxis of respiratory        diseases and conditions.    -   The use of a compound of general formula (I) in the preparation        of a medicament for the treatment or prophylaxis of skin        conditions or ocular conditions

There is also provided:

-   -   A method for the treatment or prophylaxis of respiratory        diseases and conditions, the method comprising administering to        a patient in need of such treatment an effective amount of a        compound of general formula (I).    -   A method for the treatment or prophylaxis of skin conditions and        ocular conditions, the method comprising administering to a        patient in need of such treatment an effective amount of a        compound of general formula (I).

Respiratory diseases and conditions which may be treated by thecompounds of general formula (I) include cystic fibrosis, chronicobstructive pulmonary disease (COPD), chronic bronchitis, emphysema,bronchiectasis, including non-cystic fibrosis bronchiectasis, asthma andprimary ciliary dyskinesia.

Skin conditions which may be treated by the compounds of the presentinvention include psoriasis, atopic dermatitis and ichthyosis.

Ocular conditions which may be treated by the compounds of the presentinvention included dry eye disease.

Compounds of the present invention have good ENaC blocking activity.They are particularly suitable for treating respiratory diseases becausethey have a prolonged retention time in the lungs. Furthermore, in vivoexperiments have shown that treatment with compounds of the presentinvention significantly increases mucocilliary clearance.

The patient to be treated is suitably a mammal and more suitably ahuman.

The compounds of general formula (I) may be administered in apharmaceutical composition and therefore in a further aspect of theinvention there is provided a pharmaceutical composition comprising acompound of general formula (I) and a pharmaceutically acceptableexcipient. Other pharmacologically active materials may also be present,as considered appropriate or advisable for the disease or conditionbeing treated or prevented.

The carrier, or, if more than one be present, each of the carriers, mustbe acceptable in the sense of being compatible with the otheringredients of the formulation and not deleterious to the recipient.

The formulations include those suitable for oral, rectal, nasal,bronchial (inhaled), topical (including dermal, transdermal, eye drops,buccal and sublingual), vaginal or parenteral (including subcutaneous,intramuscular, intravenous and intradermal) administration and may beprepared by any methods well known in the art of pharmacy.

The route of administration will depend upon the condition to be treatedbut preferred compositions are formulated for oral, nasal, bronchial ortopical administration.

The composition may be prepared by bringing into association the abovedefined active agent with the carrier. In general, the formulations areprepared by uniformly and intimately bringing into association theactive agent with liquid carriers or finely divided solid carriers orboth, and then if necessary shaping the product. The invention extendsto methods for preparing a pharmaceutical composition comprisingbringing a compound of general formula (I) in conjunction or associationwith a pharmaceutically acceptable carrier or vehicle.

Formulations for oral administration in the present invention may bepresented as: discrete units such as capsules, sachets or tablets eachcontaining a predetermined amount of the active agent; as a powder orgranules; as a solution or a suspension of the active agent in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water in oil liquid emulsion; or as a bolus etc.

For compositions for oral administration (e.g. tablets and capsules),the term “acceptable carrier” includes vehicles such as commonexcipients e.g. binding agents, for example syrup, acacia, gelatin,sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose,ethylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers,for example corn starch, gelatin, lactose, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride andalginic acid; and lubricants such as magnesium stearate, sodium stearateand other metallic stearates, glycerol stearate, stearic acid, siliconefluid, talc waxes, oils and colloidal silica. Flavouring agents such aspeppermint, oil of wintergreen, cherry flavouring and the like can alsobe used. It may be desirable to add a colouring agent to make the dosageform readily identifiable. Tablets may also be coated by methods wellknown in the art.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active agent in a free flowingform such as a powder or granules, optionally mixed with a binder,lubricant, inert diluent, preservative, surface-active or dispersingagent. Moulded tablets may be made by moulding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.The tablets may optionally be coated or scored and may be formulated soas to provide slow or controlled release of the active agent.

Other formulations suitable for oral administration include lozengescomprising the active agent in a flavoured base, usually sucrose andacacia or tragacanth; pastilles comprising the active agent in an inertbase such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the active agent in a suitable liquid carrier.

More suitably a compound of formula (I) is administered topically to thelung, eye or skin. Hence there is provided according to the invention apharmaceutical composition comprising a compound of the general formula(I) optionally in combination with one or more topically acceptablediluents or carriers.

For topical application to the skin, compounds of general formula (I)may be made up into a cream, ointment, jelly, solution or suspensionetc. Cream or ointment formulations that may be used for the drug areconventional formulations well known in the art, for example, asdescribed in standard text books of pharmaceutics such as the BritishPharmacopoeia.

Topical administration to the lung may be achieved by use of an aerosolformulation. Aerosol formulations typically comprise the activeingredient suspended or dissolved in a suitable aerosol propellant, suchas a chlorofluorocarbon (CFC) or a hydrofluorocarbon (HFC). Suitable CFCpropellants include trichloromonofluoromethane (propellant 11),dichlorotetrafluoromethane (propellant 114), and dichlorodifluoromethane(propellant 12). Suitable HFC propellants include tetrafluoroethane(HFC-134a) and heptafluoropropane (HFC-227). The propellant typicallycomprises 40%-99.5% e.g. 40%-90% by weight of the total inhalationcomposition. The formulation may comprise excipients includingco-solvents (e.g. ethanol) and surfactants (e.g. lecithin, sorbitantrioleate and the like). Other possible excipients include polyethyleneglycol, polyvinylpyrrolidone, glycerine and the like. Aerosolformulations are packaged in canisters and a suitable dose is deliveredby means of a metering valve (e.g. as supplied by Bespak, Valois or 3Mor alternatively by Aptar, Coster or Vari).

Topical administration to the lung may also be achieved by use of anon-pressurised formulation such as an aqueous solution or suspension.These may be administered by means of a nebuliser e.g. one that can behand-held and portable or for home or hospital use (ie non-portable).The formulation may comprise excipients such as water, buffers, tonicityadjusting agents, pH adjusting agents, surfactants and co-solvents.Suspension liquid and aerosol formulations (whether pressurised orunpressurised) will typically contain the compound of the invention infinely divided form, for example with a D₅₀ of 0.5-10 μm e.g. around 1-5μm. Particle size distributions may be represented using D₁₀, D₅₀ andD₉₀ values. The D₅₀ median value of particle size distributions isdefined as the particle size in microns that divides the distribution inhalf. The measurement derived from laser diffraction is more accuratelydescribed as a volume distribution, and consequently the D₅₀ valueobtained using this procedure is more meaningfully referred to as a Dv₅₀value (median for a volume distribution). As used herein Dv values referto particle size distributions measured using laser diffraction.Similarly, D₁₀ and D₉₀ values, used in the context of laser diffraction,are taken to mean Dv₁₀ and Dv₉₀ values and refer to the particle sizewhereby 10% of the distribution lies below the D₁₀ value, and 90% of thedistribution lies below the D₉₀ value, respectively.

Topical administration to the lung may also be achieved by use of adry-powder formulation. A dry powder formulation will contain thecompound of the disclosure in finely divided form, typically with a massmean diameter (MMAD) of 1-10 μm or a D₅₀ of 0.5-10 μm e.g. around 1-5μm. Powders of the compound of the invention in finely divided form maybe prepared by a micronization process or similar size reductionprocess. Micronization may be performed using a jet mill such as thosemanufactured by Hosokawa Alpine. The resultant particle sizedistribution may be measured using laser diffraction (e.g. with aMalvern Mastersizer 2000S instrument). The formulation will typicallycontain a topically acceptable diluent such as lactose, glucose ormannitol (preferably lactose), usually of comparatively large particlesize e.g. a mass mean diameter (MMAD) of 50 μm or more, e.g. 100 μm ormore or a D₅₀ of 40-150 μm. As used herein, the term “lactose” refers toa lactose-containing component, including α-lactose monohydrate,β-lactose monohydrate, α-lactose anhydrous, β-lactose anhydrous andamorphous lactose. Lactose components may be processed by micronization,sieving, milling, compression, agglomeration or spray drying.Commercially available forms of lactose in various forms are alsoencompassed, for example Lactohale® (inhalation grade lactose; DFEPharma), InhaLac®70 (sieved lactose for dry powder inhaler; Meggle),Pharmatose® (DFE Pharma) and Respitose® (sieved inhalation gradelactose; DFE Pharma) products. In one embodiment, the lactose componentis selected from the group consisting of α-lactose monohydrate,α-lactose anhydrous and amorphous lactose. Preferably, the lactose isα-lactose monohydrate.

Dry powder formulations may also contain other excipients. Thus in oneembodiment a dry powder formulation according the present disclosurecomprises magnesium or calcium stearate. Such formulations may havesuperior chemical and/or physical stability especially when suchformulations also contain lactose.

A dry powder formulation is typically delivered using a dry powderinhaler (DPI) device. Example dry powder delivery systems includeSPINHALER®, DISKHALER®, TURBOHALER®, DISKUS®, SKYEHALER®, ACCUHALER® andCLICKHALER®. Further examples of dry powder delivery systems includeECLIPSE, NEXT, ROTAHALER, HANDIHALER, AEROLISER, CYCLOHALER,BREEZHALER/NEOHALER, MONODOSE, FLOWCAPS, TWINCAPS, X-CAPS, TURBOSPIN,ELPENHALER, MIATHALER, TWISTHALER, NOVOLIZER, PRESSAIR, ELLIPTA, ORIELdry powder inhaler, MICRODOSE, PULVINAL, EASYHALER, ULTRAHALER, TAIFUN,PULMOJET, OMNIHALER, GYROHALER, TAPER, CONIX, XCELOVAIR and PROHALER.

In one embodiment a compound of general formula (I) is provided as amicronized dry powder formulation, for example comprising lactose of asuitable grade.

Thus, as an aspect of the invention there is provided a pharmaceuticalcomposition comprising a compound of general formula (I) in particulateform in combination with particulate lactose, said compositionoptionally comprising magnesium stearate.

In one embodiment a compound of general formula (I) is provided as amicronized dry powder formulation, comprising lactose of a suitablegrade and magnesium stearate, filled into a device such as DISKUS.Suitably, such a device is a multidose device, for example theformulation is filled into blisters for use in a multi-unit dose devicesuch as DISKUS.

In another embodiment a compound of general formula (I) is provided as amicronized dry powder formulation, for example comprising lactose of asuitable grade, filled into hard shell capsules for use in a single dosedevice such as AEROLISER.

In another embodiment a compound of general formula (I) is provided as amicronized dry powder formulation, comprising lactose of a suitablegrade and magnesium stearate, filled into hard shell capsules for use ina single dose device such as AEROLISER.

In another embodiment a compound of general formula (I) is provided as afine powder for use in an inhalation dosage form wherein the powder isin fine particles with a D₅₀ of 0.5-10 μm e.g. around 1-5 μm, that havebeen produced by a size reduction process other than jet millmicronisation e.g. spray drying, spray freezing, microfluidisation, highpressure homogenisation, super critical fluid crystallisation,ultrasonic crystallisation or combinations of these methods thereof, orother suitable particle formation methods known in the art that are usedto produce fine particles with an aerodynamic particle size of 0.5-10μm. The resultant particle size distribution may be measured using laserdiffraction (e.g. with a Malvern Mastersizer 2000S instrument). Theparticles may either comprise the compound alone or in combination withsuitable other excipients that may aid the processing. The resultantfine particles may form the final formulation for delivery to humans ormay optionally be further formulated with other suitable excipients tofacilitate delivery in an acceptable dosage form.

The compound of the invention may also be administered rectally, forexample in the form of suppositories or enemas, which include aqueous oroily solutions as well as suspensions and emulsions and foams. Suchcompositions are prepared following standard procedures, well known bythose skilled in the art. For example, suppositories can be prepared bymixing the active ingredient with a conventional suppository base suchas cocoa butter or other glycerides. In this case, the drug is mixedwith a suitable non-irritating excipient which is solid at ordinarytemperatures but liquid at the rectal temperature and will thereforemelt in the rectum to release the drug. Such materials are cocoa butterand polyethylene glycols.

Generally, for compositions intended to be administered topically to theeye in the form of eye drops or eye ointments, the total amount of thecompound of general formula (I) will be about 0.0001 to less than 4.0%(w/w).

Preferably, for topical ocular administration, the compositionsadministered according to general formula (I) will be formulated assolutions, suspensions, emulsions and other dosage forms. Aqueoussolutions are generally preferred, based on ease of formulation, as wellas a patient's ability to administer such compositions easily by meansof instilling one to two drops of the solutions in the affected eyes.However, the compositions may also be suspensions, viscous orsemi-viscous gels, or other types of solid or semi-solid compositions.Suspensions may be preferred for compounds that are sparingly soluble inwater.

An alternative for administration to the eye is intravitreal injectionof a solution or suspension of the compound of general formula (I). Inaddition, the compound of general formula (I) may also be introduced bymeans of ocular implants or inserts.

The compositions administered according to general formula (I) may alsoinclude various other ingredients, including, but not limited to,tonicity agents, buffers, surfactants, stabilizing polymer,preservatives, co-solvents and viscosity building agents. Suitablepharmaceutical compositions of general formula (I) include a compound ofthe invention formulated with a tonicity agent and a buffer. Thepharmaceutical compositions of general formula (I) may furtheroptionally include a surfactant and/or a palliative agent and/or astabilizing polymer.

Various tonicity agents may be employed to adjust the tonicity of thecomposition, preferably to that of natural tears for ophthalmiccompositions. For example, sodium chloride, potassium chloride,magnesium chloride, calcium chloride, simple sugars such as dextrose,fructose, galactose, and/or simply polyols such as the sugar alcoholsmannitol, sorbitol, xylitol, lactitol, isomaltitol, maltitol, andhydrogenated starch hydrolysates may be added to the composition toapproximate physiological tonicity. Such an amount of tonicity agentwill vary, depending on the particular agent to be added. In general,however, the compositions will have a tonicity agent in an amountsufficient to cause the final composition to have an ophthalmicallyacceptable osmolality (generally about 150-450 mOsm, preferably 250-350mOsm and most preferably at approximately 290 mOsm). In general, thetonicity agents of the invention will be present in the range of 2 to 4%w/w. Preferred tonicity agents of the invention include the simplesugars or the sugar alcohols, such as D-mannitol.

An appropriate buffer system (e.g. sodium phosphate, sodium acetate,sodium citrate, sodium borate or boric acid) may be added to thecompositions to prevent pH drift under storage conditions. Theparticular concentration will vary, depending on the agent employed.Preferably however, the buffer will be chosen to maintain a target pHwithin the range of pH 5 to 8, and more preferably to a target pH of pH5 to 7.

Surfactants may optionally be employed to deliver higher concentrationsof compound of general formula (I). The surfactants function tosolubilise the compound and stabilise colloid dispersion, such asmicellar solution, microemulsion, emulsion and suspension. Examples ofsurfactants which may optionally be used include polysorbate, poloxamer,polyosyl 40 stearate, polyoxyl castor oil, tyloxapol, Triton, andsorbitan monolaurate. Preferred surfactants to be employed in theinvention have a hydrophile/lipophile/balance “HLB” in the range of 12.4to 13.2 and are acceptable for ophthalmic use, such as TritonX114 andtyloxapol.

Additional agents that may be added to the ophthalmic compositions ofcompounds of general formula (I) are demulcents which function as astabilising polymer. The stabilizing polymer should be an ionic/chargedexample with precedence for topical ocular use, more specifically, apolymer that carries negative charge on its surface that can exhibit azeta-potential of (−)10-50 mV for physical stability and capable ofmaking a dispersion in water (i.e. water soluble). A preferredstabilising polymer of the invention would be polyelectrolyte, orpolyelectrolytes if more than one, from the family of cross-linkedpolyacrylates, such as carbomers and Pemulen®, specifically Carbomer974p (polyacrylic acid), at 0.1-0.5% w/w.

Other compounds may also be added to the ophthalmic compositions of thecompound of general formula (I) to increase the viscosity of thecarrier. Examples of viscosity enhancing agents include, but are notlimited to: polysaccharides, such as hyaluronic acid and its salts,chondroitin sulfate and its salts, dextrans, various polymers of thecellulose family; vinyl polymers; and acrylic acid polymers.

Topical ophthalmic products are typically packaged in multidose form.Preservatives are thus required to prevent microbial contaminationduring use. Suitable preservatives include: benzalkonium chloride,chlorobutanol, benzododecinium bromide, methyl paraben, propyl paraben,phenylethyl alcohol, edentate disodium, sorbic acid, polyquaternium-1,or other agents known to those skilled in the art. Such preservativesare typically employed at a level of from 0.001 to 1.0% w/v. Unit dosecompositions of general formula (I) will be sterile, but typicallyunpreserved. Such compositions, therefore, generally will not containpreservatives.

The medical practitioner, or other skilled person, will be able todetermine a suitable dosage for the compound of general formula (I), andhence the amount of the compound of the invention that should beincluded in any particular pharmaceutical formulation (whether in unitdosage form or otherwise).

Compounds of general formula (I) may be used in combination with one ormore other active agents which are useful in the treatment orprophylaxis of respiratory diseases and conditions.

An additional active agent of this type may be included in thepharmaceutical composition described above but alternatively it may beadministered separately, either at the same time as the compound ofgeneral formula (I) or at an earlier or later time.

Therefore, in a further aspect of the present invention there isprovided a product comprising a compound of general formula (I) and anadditional agent useful in the treatment or prevention of respiratoryconditions as a combined preparation for simultaneous, sequential orseparate use in the treatment of a disease or condition mediated by ENaCand especially a respiratory disease or condition, for example one ofthe diseases and conditions mentioned above.

There is also provided a compound of general formula (I) in combinationwith an additional agent useful in the treatment or prevention ofrespiratory conditions as a combined preparation for simultaneous,sequential or separate use in the treatment of a disease or conditionmediated by ENaC and especially a respiratory disease or condition, forexample one of the diseases and conditions mentioned above.

Suitable additional active agents which may be included in apharmaceutical composition or a combined preparation with the compoundsof general formula (I) include:

-   -   β2 adrenoreceptor agonists such as metaproterenol,        isoproterenol, isoprenaline,    -   albuterol, salbutamol, formoterol, salmeterol, indacaterol,        terbutaline, orciprenaline, bitolterol mesylate and pirbuterol;    -   antihistamines, for example histamine H₁ receptor antagonists        such as loratadine, cetirizine, desloratadine, levocetirizine,        fexofenadine, astemizole, azelastine and chlorpheniramine or H₄        receptor antagonists;    -   dornase alpha;    -   corticosteroids such as prednisone, prednisolone, flunisolide,        triamcinolone acetonide, beclomethasone dipropionate,        budesonide, fluticasone propionate mometasone furoate and        fluticasone furoate;    -   leukotriene antagonists such as montelukast and zafirlukast;    -   CFTR repair therapies e.g. CFTR potentiators such as Ivacaftor        and CFTR correctors such as Lumacaftor and Tezacaftor;    -   TMEM16A modulators, particularly TMEM16A potentiators;    -   antibiotics.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in greater detail with reference tothe non-limiting examples and to the drawings in which:

FIG. 1 is a plot showing the results of the sheep MCC experiment ofExample 70 showing clearance of 99mTc-SC from the lungs of sheep treatedwith the compound of Example 14 at doses of 107 μg/kg (▴) and 250 μg/kg(●) and water (▪).

FIG. 2 is a plot showing the results of the sheep MCC experiment ofExample 70 showing clearance of 99mTc-SC from the lungs of sheep treatedwith the compound of Example 4 at doses of 158 μg/kg (●) compared withwater (▪).

FIG. 3 is a series of two plots showing the results of the sheep MCCexperiment of Example 70 showing clearance of 99mTc-SC from the lungs ofsheep treated with the compound of Example 50; A—Compound 50 at doses of6 μg/kg (●) 13 μg/kg (▴) and 24 μg/kg (♦) and water (▪); B—Repeat dosesof Compound 50 at 3 μg/kg BiD (●) and 13 μg/kg (▴) and water (▪).

FIG. 4 is a plot showing the results of the sheep MCC experiment ofExample 70 showing clearance of 99mTc-SC from the lungs of sheep treatedwith the compound of Example 47 at a dose of 28 μg/kg (●) compared withwater (▪).

FIG. 5 is a plot showing the results of the sheep MCC experiment ofExample 70 showing clearance of 99mTc-SC from the lungs of sheep treatedwith the compound of Example 48 at a dose of 26 μg/kg (●) compared withwater (▪).

FIG. 6 is a plot showing the results of the sheep MCC experiment ofExample 70 showing clearance of 99mTc-SC from the lungs of sheep treatedwith the compound of Example 49 at a dose of 31 μg/kg (●) compared withwater (▪).

FIG. 7 is a plot showing the results of the sheep MCC experiment ofExample 70 showing clearance of 99mTc-SC from the lungs of sheep treatedwith the compound of Example 45 at a dose of 26 μg/kg (●) compared withwater (▪).

FIG. 8 is a plot showing the results of the sheep MCC experiment ofExample 70 showing clearance of 99mTc-SC from the lungs of sheep treatedwith the compound of Example 46 at a dose of 25 μg/kg (●) compared withwater (▪).

FIG. 9 is a plot showing the results of the sheep MCC experiment ofExample 70 showing clearance of 99mTc-SC from the lungs of sheep treatedwith the compound of Example 51 at a dose of 25 μg/kg (●) compared withwater (▪).

FIG. 10 is a plot showing the results of the sheep MCC experiment ofExample 70 showing clearance of 99mTc-SC from the lungs of sheep treatedwith the compound of Example 31 at a dose of 25 μg/kg (●) compared withwater (▪).

FIG. 11 is a plot showing the results of the sheep MCC experiment ofExample 70 showing clearance of 99mTc-SC from the lungs of sheep treatedwith the compound of Example 41 at a dose of 25 μg/kg (●) compared withwater (▪).

FIG. 12 is a plot showing the results of the sheep MCC experiment ofExample 70 showing clearance of 99mTc-SC from the lungs of sheep treatedwith the compound of Example 52 at a dose of 15 μg/kg (●) compared withwater (▪).

FIG. 13 is a plot showing the results of the sheep MCC experiment ofExample 70 showing clearance of 99mTc-SC from the lungs of sheep treatedwith the compound of Example 44 at a dose of 19 μg/kg (●) compared withwater (▪).

EXAMPLES

All reactions involving moisture-sensitive reagents were carried outunder a nitrogen atmosphere using standard vacuum line techniques andoven-dried glassware. Commercial anhydrous solvents were used inreactions and HPLC grade solvents were employed for work-up andchromatography. Water was purified using an Elix UV-5 system. All otherreagents were used as supplied without prior purification. Reportedyields are corrected for LC/MS purity (determined by UV (215 nm) or ELSdetection) unless otherwise stated. Sealed tube reactions were carriedout in heavy wall Ace pressure tubes. Microwave experiments were carriedout using a Biotage Initiator+in crimp-sealed Biotage microwave vials.Flash column chromatography was carried out using a Biotage Isolera 4using Biotage SNAP KP or SNAP Ultra columns. NMR spectra were recordedon a Bruker Avance III HD 500 MHz or a Bruker Avance III HD 250 MHzusing the solvent as internal deuterium lock. Spectra were recorded atroom temperature unless otherwise stated.

Analytical LC/MS were carried out on the following systems:

-   -   System A: stationary phase: Kinetex Core-Shell C18 2.1×50 mm, 5        μm, 40° C.; detection UV 215 nm-ELS-MS; mobile phase: A,        water+0.1% formic acid; B, MeCN+0.1% formic acid; gradient (A:B        ratio, time): 95:5-0:100, 1.20 min; 100:0, 0.10 min; 100:0-5:95,        0.01 min; flowrate: 1.2 ml/min;    -   System B: stationary phase: Phenomenex Gemini-NX C18 2.0×50 mm,        3 μm, 60° C.; detection UV 215 nm-ELS-MS; mobile phase: A, 2 mM        ammonium bicarbonate pH 10; B, MeCN; gradient (A:B ratio, time):        99:1-0:100, 1.80 min; 100:0, 0.30 min; 100:0-1:99, 0.20 min;        1:99, 1.20 min; flowrate: 1.0 ml/min;    -   System C: stationary phase: Phenomenex Kinetex-XB C18 2.1×100        mm, 1.7 μm, 40° C.; detection UV 215 nm-ELS-MS; mobile phase: A,        water+0.1% formic acid; B, MeCN+0.1% formic acid; gradient (A:B        ratio, time): 95:5-0:100, 5.30 min; 100:0, 0.50 min; 100:0-5:95,        0.02 min; 5:95, 1.18 min; flowrate: 0.6 ml/min;    -   System D: stationary phase: Waters CSH C18 2.1×100 mm, 1.7 μm,        40° C.; detection UV 215 nm-ELS-MS; mobile phase: A, 5 mM        ammonium acetate pH 7; B, MeCN; gradient (A:B ratio, time):        95:5-0:100, 5.30 min; 100:0, 0.50 min; 100:0-5:95, 0.02 min;        5:95, 1.18 min; flowrate: 0.6 ml/min.

Preparative HPLC purification was carried out using the followingconditions:

-   -   Method A: stationary phase: Waters Sunfire 30×100 mm, 10 μm;        detection UV 215 and 254 nm; mobile phase A: water+0.1% formic        acid; B: MeCN+0.1% formic acid; gradient: 5-95% solvent B over        14 min; flowrate: 40 ml/min;    -   Method B: stationary phase: Waters Sunfire 30×100 mm, 5 μm;        detection UV 215 and 254 nm; mobile phase A: water+0.1% formic        acid; B: MeCN+0.1% formic acid; gradient: 2-12% solvent B over        12 min; flowrate: 40 ml/min;    -   Method C: stationary phase: XSelect CSH C18 30×100 mm, 5 μm;        detection UV 220 nm; mobile phase A: water+0.1% TFA; B:        MeCN+0.1% TFA; gradient: 5-15% solvent B over 21 min; flowrate:        42 ml/min;    -   Method D: stationary phase: XSelect CSH C18 30×100 mm, 5 μm;        detection UV 220 nm; mobile phase A: water+0.1% TFA; B:        MeCN+0.1% TFA; gradient: 2-15% solvent B over 12 min; flowrate:        42 ml/min;    -   Method E: stationary phase: XSelect CSH C18 30×100 mm, 5 μm;        detection UV 220 nm; mobile phase A: water+0.1% TFA; B:        MeCN+0.1% TFA; gradient: 10-35% solvent B over 10 min; flowrate:        42 ml/min.

The following abbreviations and terms have the indicated meaningsthroughout:

-   -   AcOH glacial acetic acid    -   br broad    -   CDI 1,1′-carbonyldiimidazole    -   CV column volumes    -   dd doublet of doublets    -   DIPEA N,N-diisopropylethylamine    -   DMF N,N-dimethylformamide    -   DMSO dimethyl sulfoxide    -   dppf 1,1′-bis(diphenylphosphino)ferrocene    -   EDC·HCl N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide        hydrochloride    -   ELS evaporative light scattering    -   ESI electrospray ionisation    -   EtOAc ethyl acetate    -   FMOC fluorenylmethyloxycarbonyl    -   HATU        1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium        3-oxide hexafluorophosphate    -   HBTU 3-[bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide        hexafluorophosphate    -   HOAt 1-hydroxy-7-azabenzotriazole    -   HPLC high-performance liquid chromatography    -   LC/MS liquid chromatography-mass spectrometry    -   m multiplet    -   MeCN acetonitrile    -   MeOH methanol    -   NCS N-chlorosuccinimide    -   NMR nuclear magnetic resonance    -   Ph phenyl    -   q quartet    -   RT room temperature    -   Rt retention time    -   s singlet    -   SCX strong cation exchange    -   SPhos 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl    -   t triplet    -   t-Bu tert-butyl    -   TBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium        tetrafluoroborate    -   TFA trifluoroacetic acid    -   THF tetrahydrofuran    -   XPhos-Pd-G2        chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II)

A. SYNTHESIS OF INTERMEDIATES Intermediate 1—Synthesis of methyl3,5-diamino-6-[(E)-2-ethoxyethenyl]pyrazine-2-carboxylate

A mixture of methyl 3,5-diamino-6-chloropyrazine-2-carboxylate (40.0 g,197 mmol),2-[(E)-2-ethoxyethenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (84 ml,400 mmol), SPhos (8.11 g, 19.7 mmol), palladium(II) acetate (2.22 g,9.87 mmol) and K₃PO₄ (83.8 g, 395 mmol) in water:MeCN (2:3, 350 ml) wasstirred at 80° C. for 2 h then allowed to cool to RT. The solid wascollected by filtration then washed with EtOAc (100 ml) and water (100ml) then dried in vacuo to afford the product as a brown solid (36.7 g,76%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.15 (d, J=12.2 Hz, 1H), 6.79 (s, 4H), 5.97(d, J=12.2 Hz, 1H), 3.91 (q, J=7.0 Hz, 2H), 3.72 (s, 3H), 1.25 (t, J=7.0Hz, 3H).

LC/MS (System A): m/z (ESI+)=239 [MH⁺], Rt=0.88 min, UV purity=98%.

Intermediate 2—Synthesis of methyl3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate

A solution of methyl3,5-diamino-6-[(E)-2-ethoxyethenyl]pyrazine-2-carboxylate, Intermediate1 (98%, 36.6 g, 151 mmol) in AcOH (360 ml) was heated at 80° C. for 16h. The reaction was allowed to cool to RT then concentrated in vacuo.The residue was azeotroped with toluene (2×100 ml). The resultingresidue was dissolved in CH₂Cl₂:MeOH (4:1, 600 ml) then warmed at 50° C.Activated charcoal (5 g) was added then the resultant mixture wasstirred at 50° C. for 0.5 h. The mixture was allowed to cool thenfiltered through a Celite pad. The filtrate was concentrated in vacuo toafford the product as a brown solid (20.2 g, 68%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.48 (s, 1H), 7.50 (d, J=3.8 Hz, 1H), 7.05(s, 2H), 6.42 (d, J=3.7 Hz, 1H), 3.84 (s, 3H).

LC/MS (System A): m/z (ESI+)=193 [MH⁺], Rt=0.76 min, UV purity=98%.

Intermediate 3—Synthesis of lithium (1⁺) ion3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate

Aqueous LiOH solution (1.0 M, 210 ml, 210 mmol) was added to a mixtureof methyl 3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate2 (20.2 g, 105 mmol) in MeOH (300 ml). The resulting mixture was heatedat 50° C. for 16 h then allowed to cool to RT. The solid was collectedby filtration then washed with water and MeOH and then dried undervacuum to afford the product as a yellow solid (15.6 g, 79%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.17 (s, 1H), 8.62-6.43 (m, 3H), 6.18 (d,J=3.6 Hz, 1H).

LC/MS (System C): m/z (ESI+)=179 [MH⁺], Rt=0.96 min, UV purity=97%.

Intermediate 4—Synthesis of2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine

A mixture of lithium(1⁺) ion3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate 3 (5.00 g,27.2 mmol), CDI (6.61 g, 40.7 mmol) and 1H-imidazole hydrochloride (1:1)(3.12 g, 29.9 mmol) in DMF (80 ml) was stirred at RT for 16 h.Additional CDI (2.00 g, 12.3 mmol) was added and the reaction was leftto stir for a further 1 h at RT. The reaction mixture was diluted withwater (350 ml) then stirred at RT for 5 min then left to stand at RT for0.5 h. The resultant mixture was filtered then the collected solid waswashed with water then dried under vacuum to afford the product as ayellow/orange solid (3.30 g, 52%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.69 (s, 1H), 8.74 (t, J=0.9 Hz, 1H), 7.94(t, J=1.4 Hz, 1H), 7.63 (dd, J=3.8, 2.3 Hz, 1H), 7.44 (s, 2H), 7.08 (dd,J=1.5, 0.8 Hz, 1H), 6.52 (dd, J=3.8, 1.3 Hz, 1H).

LC/MS (System A): m/z (ESI+)=229 [MH⁺], Rt=0.65 min, UV purity=97%.

Intermediate 7—Synthesis of methyl3,5-diamino-6-[3-(trimethylsilyl)prop-1-yn-1-yl]pyrazine-2-carboxylate

Trimethyl(prop-2-yn-1-yl)silane (332 mg, 2.96 mmol) was added to asolution of methyl 3,5-diamino-6-chloropyrazine-2-carboxylate (500 mg,2.47 mmol), PdCl₂(PPh3)₂ (170 mg, 0.25 mmol), CuI (47 mg, 0.25 mmol) andtriethylamine (520 μl, 3.7 mmol) in DMSO (5 ml) in a pressure tube. Thetube was sealed then heated at 60° C. for 2 h. The reaction was allowedto cool to RT then left to stir at RT for 16 h, then at 60° C. again fora further 2 h. The reaction mixture was diluted with EtOAc (20 ml) andwater (20 ml) then filtered through a Celite pad. The phases of thefiltrate were separated then the organic phase was extracted with water(2×50 ml) and brine (100 ml), then dried over Na₂SO₄ and concentrated invacuo to a viscous orange/brown oil. The crude material was purified byflash column chromatography on a silica column (50 g). The column waseluted with EtOAc:heptane, using the following gradient (% EtOAc, columnvolumes): 0%, 1 CV; 0-46%, 6 CV; 46-67%, 2 CV; 67%, 1 CV; 67-100%, 3 CV.The desired fractions were combined and concentrated in vacuo to affordthe product as a yellow solid (330 mg, 44%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.12 (s, 4H), 3.72 (s, 3H), 1.84 (s, 2H),0.13 (s, 9H).

LC/MS (System A): m/z (ESI+)=279 [MH⁺], Rt=1.11 min, UV purity=92%.

Intermediate 8—Synthesis of methyl3-amino-6-methyl-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate

Potassium tert-butoxide (141 mg, 1.26 mmol) was added to a solution ofmethyl3,5-diamino-6-[3-(trimethylsilyl)prop-1-yn-1-yl]pyrazine-2-carboxylate,Intermediate 7 (92%, 318 mg, 1.05 mmol) in THF (25 ml). The reactionmixture was stirred under reflux for 1 h. Potassium tert-butoxide (141mg, 1.26 mmol) was added then the reaction was heated under reflux for afurther 1 h. The reaction mixture was diluted with EtOAc (100 ml) thenextracted with saturated aqueous NH₄Cl (50 ml), water (50 ml) and brine(50 ml). The organic phase was dried over Na₂SO₄ and concentrated invacuo. The crude material was purified by flash column chromatography onC18 (30 g). The column was eluted with MeCN:water+0.1% formic acid usingthe following gradient (% MeCN, column volumes): 0%, 1 CV; 0-7%, 2 CV;7-9%, 1 CV; 9-41%, 4 CV; 41-83%, 3 CV; 100% 1 CV. The desired fractionswere combined and concentrated in vacuo to afford the product as ayellow solid (105 mg, 40%).

¹H NMR (250 MHz, DMSO-d₆) δ 11.35 (s, 1H), 6.97 (s, 2H), 6.22-6.02 (m,1H), 3.81 (s, 3H), 2.35 (d, J=1.0 Hz, 3H).

LC/MS (System A): m/z (ESI+)=207 [MH⁺], Rt=0.82 min, UV purity=82%.

Intermediate 9—Synthesis of3-amino-6-methyl-5H-pyrrolo[2,3-b]pyrazine-2-carboxylic acid

Aqueous LiOH solution (1.0 M, 1.0 ml, 1.0 mmol) was added to a solutionof methyl 3-amino-6-methyl-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate,Intermediate 8 (82%, 100 mg, 0.40 mmol) in MeOH (4 ml). The reactionmixture was stirred at 50° C. for 1 h. Aqueous LiOH solution (1.0 M, 1.0ml, 1.0 mmol) was added then the reaction mixture was stirred at 65° C.for a further 1 h. The reaction mixture was allowed to cool thenconcentrated in vacuo. The resulting residue was dissolved in water (5ml) then acidified to pH 3 using dilute aqueous HCl solution. Themixture was left to stand at RT for 64 h then filtered. The resultingsolid was washed with water (2 ml) then dried under vacuum to afford theproduct as a brown solid (85 mg, >99%).

¹H NMR (250 MHz, DMSO-d₆) δ 11.39 (s, 1H), 6.91 (br. s, 2H), 6.14 (s,1H), 2.36 (s, 3H).

LC/MS (System A): m/z (ESI+)=193 [MH⁺], Rt=0.64 min, UV purity=90%.

Intermediate 10—Synthesis of methyl3-amino-7-iodo-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate

A mixture of methyl 3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate,Intermediate 2 (98%, 1.50 g, 7.65 mmol) and NIS (1.84 g, 8.16 mmol) inacetone (30 ml) was stirred at RT for 2 h. Additional NIS (350 mg, 1.56mmol) was added and the reaction was stirred at RT for a further 0.5 h.The reaction mixture was concentrated in vacuo to a black solid. Thecrude material was purified by flash column chromatography on a silicacolumn (100 g). The column was eluted with CH₂Cl₂:MeOH, using thefollowing gradient (% MeOH, column volumes): 0%, 1 CV; 0-3.5%, 4 CV;3.5%, 2 CV; 3.5-4.4%, 1 CV. The desired fractions were combined andconcentrated in vacuo. The material thus obtained was further purifiedby preparative HPLC (Method A). The desired fractions were combined andconcentrated in vacuo to afford the product as a yellow/orange solid(591 mg, 24%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.89 (s, 1H), 7.72 (s, 1H), 7.21 (s, 2H),3.88 (s, 3H).

LC/MS (System A): m/z (ESI+)=319 [MH⁺], Rt=0.94 min, UV purity=100%.

Intermediate 11—Synthesis of methyl3-amino-7-methyl-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate

Dimethylzinc solution in toluene (2.0 M, 0.88 ml, 1.8 mmol) was addeddropwise over 2 min to a cooled (0° C.) suspension of methyl3-amino-7-iodo-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate 10(280 mg, 0.88 mmol) and Pd(Pt-Bu₃)₂ (45 mg, 0.09 mmol) in THF (5 ml).The reaction mixture was stirred at 0° C. for 30 min then allowed towarm to RT. Additional dimethylzinc solution in toluene (2.0 M, 0.88 ml,1.8 mmol) and a solution of Pd(Pt-Bu₃)₂ (45 mg, 0.09 mmol) in THF (2 ml)were added then the resulting mixture was stirred at RT for 10 min.Saturated aqueous NaHCO₃ solution (10 ml) was added followed by EtOAc(30 ml). The mixture was filtered through a Celite pad then the phaseswere separated. The organic phase was extracted with water (3×80 ml) andbrine (80 ml) then dried over Na₂SO₄ and concentrated in vacuo. Thecrude material was purified by flash column chromatography on a silicacolumn (100 g). The column was eluted with EtOAc:heptane, increasing thegradient linearly from 0-100% EtOAc over 10 CV. The desired fractionswere combined and concentrated in vacuo to afford the product as ayellow solid (85 mg, 46%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.13 (s, 1H), 7.25 (dd, J=2.3, 1.2 Hz, 1H),7.03 (s, 2H), 3.85 (s, 3H), 2.17 (d, J=1.2 Hz, 3H).

LC/MS (System A): m/z (ESI+)=207 [MH⁺], Rt=0.85 min, UV purity=99%.

Intermediate 12—Synthesis of lithium (1⁺) ion3-amino-7-methyl-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate

Aqueous LiOH solution (1.0 M, 1.0 ml, 1.0 mmol) was added to a mixtureof methyl 3-amino-7-methyl-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate,Intermediate 11 (110 mg, 0.53 mmol) in MeOH (1 ml). The reaction washeated at 50° C. for 1 h then allowed to cool to RT. The resultantmixture was filtered then the solid was washed with water and driedunder vacuum to afford the product as a yellow solid (91 mg, 84%).

¹H NMR (500 MHz, DMSO-d₆) δ 10.80 (s, 1H), 8.68-6.32 (m, 3H), 2.12 (s,3H).

LC/MS (System A): m/z (ESI+)=193 [MH⁺], Rt=0.77 min, UV purity=98%.

Intermediate 13—Synthesis of2-(1H-imidazole-1-carbonyl)-7-methyl-5H-pyrrolo[2,3-b]pyrazin-3-amine

A mixture of lithium(1⁺) ion3-amino-7-chloro-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate12 (90 mg, 0.45 mmol), CDI (110 mg, 0.67 mmol) and 1H-imidazolehydrochloride (1:1) (56 mg, 0.53 mmol) in DMF (2 ml) was stirred at RTfor 1 h. Additional CDI (50 mg, 0.31 mmol) was added and the reactionwas allowed to continue for a further 1 h. The reaction mixture wasdiluted with water (4 ml) then filtered. The solid was washed with waterthen dried under vacuum to afford the product as a yellow solid (83 mg,75%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.37 (s, 1H), 8.84 (s, 1H), 8.00 (t, J=1.4Hz, 1H), 7.45 (s, 2H), 7.41-7.37 (m, 1H), 7.12-7.08 (m, 1H), 2.22 (d,J=1.1 Hz, 3H).

LC/MS (System A): m/z (ESI+)=243 [MH⁺], Rt=0.78 min, UV purity=98%.

Intermediate 14—Synthesis of methyl3-amino-7-chloro-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate

A mixture of methyl 3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate,Intermediate 2 (98%, 1.00 g, 5.10 mmol) and NCS (0.561 g, 4.20 mmol) inDMF (15 ml) was stirred at 40° C. for 2 h. Additional NCS (50 mg, 0.37mmol) was added and the reaction was stirred at 40° C. for an additional2 h. The reaction mixture was partitioned between water (100 ml) andEtOAc (100 ml). The phases were separated then the organic phase waswashed with water (2×100 ml) then dried over Na₂SO₄ and concentrated invacuo. The crude material was purified by flash column chromatography ona silica column (100 g). The column was eluted with CH₂Cl₂:MeOH,increasing the gradient linearly from 100:0 to 90:10 over 8 columnvolumes then isocratic at 90:10 for 5 column volumes. The desiredfractions were combined and evaporated. The residue thus obtained wassuspended in CH₂Cl₂:MeOH (9:1, 20 ml) then filtered. The filtrate wasfurther purified by flash column chromatography on C18 (60 g). Thecolumn was eluted with MeCN:water+0.1% formic acid using the followinggradient (% MeCN, column volumes): 10%, 2 CV; 10-20%, 8 CVs; 20-37%, 8CV; 37-100%, 5 CV; 100%, 1 CV. The solid from the filtration wasdissolved in DMSO:MeCN (2:1) then purified by preparative HPLC (MethodA). The desired fractions from both columns were combined and evaporatedto afford the product as a yellow solid (222 mg, 19%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.76 (s, 1H), 7.68 (s, 1H), 7.24 (s, 2H),3.87 (s, 3H).

LC/MS (System A): m/z (ESI+)=227 [MH⁺], Rt=0.88 min, UV purity=100%.

Intermediate 15—Synthesis of lithium(1⁺) ion3-amino-7-chloro-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate

Aqueous LiOH solution (1.0 M, 1.9 ml, 1.9 mmol) was added to a mixtureof methyl 3-amino-7-chloro-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate,Intermediate 14 (220 mg, 0.97 mmol) in MeOH (3 ml). The reaction mixturewas heated to 50° C. for 2 h then allowed to cool to RT. The resultingsuspension was filtered then the collected solid was washed with theminimum of MeOH and water. The resulting solid was dried under vacuum toafford the product as a yellow solid (191 mg, 87%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.37 (s, 1H), 8.39-7.10 (m, 3H).

LC/MS (System A): m/z (ESI+)=213 [MH⁺], Rt=0.81 min, UV purity=97%.

Intermediate 16—Synthesis of7-chloro-2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine

A mixture of lithium(1⁺) ion3-amino-7-chloro-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate15 (190 mg, 0.869 mmol), CDI (211 g, 1.30 mmol) and 1H-imidazolehydrochloride (1:1) (100 mg, 0.956 mmol) in DMF (4 ml) was stirred at RTfor 0.5 h. Additional CDI (50 mg, 0.31 mmol) was added and the reactionwas left to stir at RT for a further 0.5 h. The reaction mixture wasdiluted with water (6 ml) then left to stir at RT for 20 min. Theresulting suspension was filtered then the collected solid was washedwith water then dried under vacuum to afford the product as ayellow/orange solid (164 mg, 72%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.98 (s, 1H), 8.80 (s, 1H), 7.99 (t, J=1.4Hz, 1H), 7.84 (s, 1H), 7.58 (s, 2H), 7.12 (dd, J=1.5, 0.8 Hz, 1H).

LC/MS (System A): m/z (ESI+)=263 [MH⁺], Rt=0.82 min, ELS purity=100%.

Intermediate 17—Synthesis of tert-butylN-[(1-ethyl-6-fluoro-1H-1,3-benzodiazol-2-yl)methyl]carbamate

A solution of 2-{[(tert-butoxy)carbonyl]amino}acetic acid (1.70 g, 9.73mmol), HATU (4.07 g, 10.7 mmol) and DIPEA (3.39 ml, 19.5 mmol) in DMF(20 ml) was stirred at RT for 20 min. A solution of1-N-ethyl-5-fluorobenzene-1,2-diamine (1.05 g, 9.73 mmol) in THF (10 ml)was added and the resulting mixture was stirred at RT for 16 h. Thereaction mixture was poured onto saturated aqueous NaHCO₃ solution (80ml). EtOAc (100 ml) and water (50 ml) were added then the phases wereseparated. The aqueous phase was extracted with EtOAc (2×50 ml) then thecombined organic phases were washed with water (4×50 ml) and brine (50ml), then dried over Na₂SO₄, filtered and evaporated to afford theintermediate as a black oil (4 g). The intermediate was dissolved inacetic acid (30 ml) then heated at 60° C. for 4 h. The reaction mixturewas allowed to cool to RT then stirred at RT for 16 h. The resultingmixture was evaporated then the resulting residue was partitionedbetween EtOAc (150 ml) and water (100 ml). The aqueous phase wasextracted with EtOAc (50 ml) then the combined organic phases werewashed with water (4×50 ml) and brine (50 ml), then dried over Na₂SO₄,filtered and evaporated to a black solid (4 g). The solid was dissolvedin the minimum of CH₂Cl₂/MeOH then evaporated onto silica (9 g). Thecrude material was purified by flash column chromatography on a silicacolumn (120 g). The column was eluted with CH₂Cl₂:MeOH, increasing thegradient linearly from 100:0 to 95:5 over 10 column volumes. The desiredfractions were combined and evaporated to a black solid (2.9 g). Thesolid thus obtained was dissolved in EtOAc (100 ml) and extracted withsaturated aqueous sodium bicarbonate solution (3×50 ml) and water (50ml) then dried over Na₂SO₄, filtered and evaporated to a black solid(2.5 g). The solid was dissolved in the minimum of CH₂Cl₂/MeOH thenevaporated onto silica (10 g). The material was further purified byflash column chromatography on a silica column (120 g). The column waseluted with EtOAc:heptane, increasing the gradient linearly from 0:100to 100:0 over 10 column volumes. The desired fractions were combined andevaporated to afford the product as a pink solid (1.78 g, 62%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.56 (dd, J=8.8, 4.9 Hz, 1H), 7.50-7.41 (m,2H), 7.01 (ddd, J=9.9, 8.9, 2.5 Hz, 1H), 4.42 (d, J=5.9 Hz, 2H), 4.24(q, J=7.2 Hz, 2H), 1.45-1.20 (m, 12H).

LC/MS (System A): m/z (ESI⁺)=294 [MH⁺], R_(t)=0.92 min, UV purity=100%.

Intermediate 18—Synthesis of2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-6-fluoro-3-methyl-1H-1,3-benzodiazol-3-iumiodide

Iodomethane (497 μl, 7.98 mmol) was added to a suspension of tert-butylN-[(1-ethyl-6-fluoro-1H-1,3-benzodiazol-2-yl)methyl]carbamate,Intermediate 17 (780 mg, 2.66 mmol) in MeCN (12 ml) in a pressure tube.The tube was sealed then heated at 75° C. for 4 h. The reaction mixturewas allowed to cool to RT then concentrated in vacuo to afford theproduct as a pale yellow solid (1.16 g, 99%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.14-8.04 (m, 2H), 7.94 (t, J=5.2 Hz, 1H),7.68-7.56 (m, 1H), 4.73 (d, J=5.4 Hz, 2H), 4.58 (q, J=7.2 Hz, 2H), 4.08(s, 3H), 1.38 (d, J=11.2 Hz, 12H).

LC/MS (System A): m/z (ESI⁺)=308 [M⁺], R_(t)=0.87 min, UV purity=99%.

Intermediate 19—Synthesis of2-(aminomethyl)-1-ethyl-6-fluoro-3-methyl-1H-1,3-benzodiazol-3-iumhydrochloride iodide

HCl solution in dioxane (4.0 M, 3.3 ml, 13.2 mmol) was added to asolution of2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-6-fluoro-3-methyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 18 (1.16 g, 2.66 mmol) in MeCN (5 ml). The reactionwas stirred at RT for 0.5 h then concentrated in vacuo. The solid wasazeotroped with MeCN (10 ml) then dried under vacuum to yield theproduct as a yellow/green solid (870 mg, 88%).

¹H NMR (500 MHz, DMSO-d₆) δ 9.21 (s, 3H), 8.25-8.14 (m, 2H), 7.69 (td,J=9.3, 2.4 Hz, 1H), 4.76 (s, 2H), 4.70 (q, J=7.2 Hz, 2H), 4.19 (s, 3H),1.42 (t, J=7.2 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=208 [M⁺], R_(t)=0.14 min, ELS purity=100%.

Intermediate 20—Synthesis of2-[(6-chloro-1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione

A mixture of N-phthaloylglycine (2.15 g, 10.5 mmol), TBTU (3.52 g, 11.0mmol) and triethylamine (2.31 ml, 13.0 mmol) in DMF (30 ml) was stirredat RT for 45 min. A solution of 5-chloro-1-N-ethylbenzene-1,2-diamine(1.70 g, 9.96 mmol) in THF (20 ml) was added and the resulting mixturewas stirred at RT for 18 h. The reaction mixture was added to saturatedaqueous NaHCO3 solution (100 ml) which caused a pale brown solid toprecipitate from solution. The solid was filtered, washed with water anddried under vacuum. The solid thus obtained was triturated in MeCN thenfiltered and dried under vacuum to afford the intermediate as a palepink solid (5.4 g). The solid thus obtained was added portion-wise toacetic acid (30 ml). The resulting suspension was heated at 100° C. for45 min then allowed to cool to RT over 16 h. The resulting suspensionwas filtered and washed with EtOAc then dried under vacuum to afford theproduct as a pale pink solid (585 mg). The solid was suspended in MeCN(5 ml) then MeCN:water (1:1, 1 ml) was added. The resulting suspensionwas filtered then the solid was dried under vacuum to afford the productas a pale pink solid (430 mg, 13%). The filtrate was again filtered,then the solid was washed with EtOAc and dried under vacuum to afford asecond batch of product as a pale pink solid (2.00 g). The solid wassuspended in MeCN (20 ml) then MeCN:water (1:1, 5 ml) was added. Theresulting suspension was filtered then the solid was dried under vacuumto afford second batch of the product as a pale pink solid (1.33 g,38%). The two batches of product were as a suspension in MeCN thenconcentrated in vacuo and dried under vacuum to afford the product as apale pink solid (1.76 g, 51%).

¹H NMR (250 MHz, DMSO-d₆) δ 8.02-7.85 (m, 4H), 7.76 (d, J=1.9 Hz, 1H),7.49 (d, J=8.6 Hz, 1H), 7.15 (dd, J=8.6, 2.0 Hz, 1H), 5.13 (s, 2H), 4.39(q, J=7.1 Hz, 2H), 1.37 (t, J=7.2 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=340 [M(³⁵Cl)H⁺], 342 [M(³⁷Cl)H⁺],R_(t)=1.12 min, UV purity=99%.

Intermediate 21—Synthesis of6-chloro-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-3-methyl-1H-1,3-benzodiazol-3-iumiodide

A mixture of2-[(6-chloro-1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione,Intermediate 20 (600 mg, 1.77 mmol) and iodomethane (330 μl, 5.30 mmol)in MeCN (6 ml) was heated at 75° C. in a sealed tube for 1.5 h thenallowed to cool to RT. Iodomethane (165 μl, 2.65 mmol) was added thenthe reaction was heated at 80° C. for 5 h. The reaction was allowed tocool to RT then filtered to afford a solid which was washed with MeCNand dried under vacuum to afford the product as a yellow solid (644 mg,73%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.40 (d, J=1.8 Hz, 1H), 8.13 (d, J=8.9 Hz,1H), 7.91 (ddt, J=18.3, 5.8, 3.1 Hz, 4H), 7.79 (dd, J=8.9, 1.9 Hz, 1H),5.40 (s, 2H), 4.74 (q, J=7.2 Hz, 2H), 4.14 (s, 3H), 1.43 (t, J=7.2 Hz,3H).

LC/MS (System A): m/z (ESI⁺)=354 [M(³⁵Cl)⁺], 356 [M(³⁷Cl)⁺], R_(t)=0.90min, UV purity=97%.

Intermediate 22—Synthesis of2-(aminomethyl)-6-chloro-1-ethyl-3-methyl-1H-1,3-benzodiazol-3-iumiodide

Hydrazine hydrate (446 μl, 9.17 mmol) was added to a suspension of6-chloro-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-3-methyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 21 (640 mg, 1.33 mmol) in MeOH (8 ml) in a pressuretube. The tube was sealed and heated at 75° C. for 3 h then allowed tocool to RT. The resulting suspension was filtered and the solid waswashed with MeOH (10 ml). The filtrate was concentrated in vacuo toafford an orange solid, which was suspended in CH₂Cl₂ (10 ml) thenfiltered and washed through with CH₂Cl₂. The solid thus obtained wassuspended in CH₂Cl₂ (10 ml). A few drops of MeOH were added and thesuspension was sonicated. The resulting suspension was filtered then thesolid was dried under vacuum to afford the product as an off-white solid(360 mg, 77%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.31 (d, J=1.9 Hz, 1H), 8.06 (d, J=8.9 Hz,1H), 7.73 (dd, J=8.8, 1.9 Hz, 1H), 4.60 (q, J=7.2 Hz, 2H), 4.25 (s, 2H),4.06 (s, 3H), 2.52-2.10 (s, 2H+DMSO), 1.41 (t, J=7.2 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=224 [M(³⁵Cl)⁺], 226 [M(³⁷Cl)⁺], R_(t)=0.16min, ELS purity=100%.

Intermediate 23—Synthesis of2-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione

Intermediate 23 was synthesised according to literature procedures (US2015/0018314 A1).

Intermediate 24—Synthesis of2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-6-methoxy-3-methyl-1H-1,3-benzodiazol-3-iumiodide

Iodomethane (590 μl, 9.47 mmol) was added to a suspension of2-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione,Intermediate 23 (1.59 g, 4.74 mmol) in MeCN (13 ml) in a pressure tube.The mixture was heated at 80° C. for 4 h then allowed to cool to RT. Theresulting suspension was reduced to approximately half of the originalvolume under a stream of nitrogen. The solid was collected by filtrationthen washed with further MeCN (3 ml) to yield the product as a whitesolid (1.99 g, 87%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.01-7.84 (m, 5H), 7.62 (d, J=2.2 Hz, 1H),7.32 (dd, J=9.2, 2.3 Hz, 1H), 5.37 (s, 2H), 4.71 (q, J=7.1 Hz, 2H), 4.11(s, 3H), 3.92 (s, 3H), 1.42 (t, J=7.2 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=350 [M⁺], R_(t)=0.87 min, UV purity=99%.

Intermediate 25—Synthesis of2-(aminomethyl)-1-ethyl-6-methoxy-3-methyl-1H-1,3-benzodiazol-3-iumiodide

Hydrazine hydrate (1.17 ml, 24.0 mmol) was added to a suspension of2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-6-methoxy-3-methyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 24 (2.29 g, 4.80 mmol) in MeOH (25 ml). The mixturewas heated at 75° C. for 1 h. The reaction was concentrated in vacuo andthe resulting solid was suspended in CH₂Cl₂:MeOH (10:1). The solid wascollected by filtration and washed with CH₂Cl₂. The filtrate wasconcentrated in vacuo to afford the product as a pale brown solid (1.60g, 96%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.91 (d, J=9.1 Hz, 1H), 7.57 (d, J=2.3 Hz,1H), 7.26 (dd, J=9.1, 2.3 Hz, 1H), 4.58 (q, J=7.2 Hz, 2H), 4.23 (s, 2H),4.03 (s, 3H), 3.91 (s, 3H), 1.42 (t, J=7.2 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=220 [M⁺], R_(t)=0.14 min, ELS purity=100%.

Intermediate 26—Synthesis of tert-butylN-{[1-ethyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-2-yl]methyl}carbamate

Intermediate 26 was synthesised according to literature procedures (WO2009019506 A1).

Intermediate 27—Synthesis of2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-3-methyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-iumiodide

Iodomethane (381 μl, 6.12 mmol) was added to a suspension of tert-butylN-{[1-ethyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-2-yl]methyl}carbamate,Intermediate 26 (700 mg, 2.04 mmol) in MeCN (10 ml) in a pressure tube.The tube was sealed and heated at 75° C. for 8 h then allowed to cool toRT. The reaction mixture was concentrated in vacuo to afford the productas a pale yellow solid (1.01 g, >99%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.64 (s, 1H), 8.29 (d, J=8.7 Hz, 1H), 8.08(d, J=8.7 Hz, 1H), 7.98 (t, J=5.2 Hz, 1H), 4.80 (d, J=5.6 Hz, 2H), 4.73(q, J=7.2 Hz, 2H), 4.14 (s, 3H), 1.40 (m, 12H).

LC/MS (System A): m/z (ESI⁺)=358 [M⁺], R_(t)=0.91 min, UV purity=98%.

Intermediate 28—Synthesis of2-(aminomethyl)-1-ethyl-3-methyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-iumhydrochloride iodide

HCl solution in dioxane (4.0 M, 2.8 ml, 11 mmol) was added to a solutionof2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-3-methyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-iumiodide, Intermediate 27 (1.07 g, 2.21 mmol) in MeCN (5 ml). The reactionwas stirred at RT for 16 h then concentrated in vacuo to yield theproduct as an off-white solid (875 mg, 94%).

¹H NMR (500 MHz, DMSO-d₆) δ 9.14 (s, 3H), 8.74 (s, 1H), 8.38 (d, J=8.8Hz, 1H), 8.14 (dd, J=8.8, 1.3 Hz, 1H), 4.84 (d, J=5.5 Hz, 4H), 4.24 (s,3H), 1.45 (t, J=7.2 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=258 [M⁺], R_(t)=0.17 min, ELS purity=100%.

Intermediate 29—Synthesis of N-ethyl-2-nitro-5-(trifluoromethoxy)aniline

Two identical reactions were carried out in parallel as follows thencombined for work-up. Ethylamine solution in THF (2.0 M, 4.1 ml, 8.2mmol) was added to a suspension of2-chloro-1-nitro-4-(trifluoromethoxy)benzene (1.00 g, 4.14 mmol) andK₂CO₃ (1.71 g, 6.21 mmol) in THF (12 ml) in a pressure tube. The tubewas sealed then heated at 50° C. for 16 h then allowed to cool to RT.Additional ethylamine solution in THF (2.0 M, 2.1 ml, 4.2 mmol) wasadded then the reaction was heated at 50° C. for 24 h. The combinedreactions were filtered then the solid thus obtained was rinsed withEtOAc (100 ml). The combined filtrates were extracted with saturatedaqueous NaHCO₃ solution (2×100 ml), water (50 ml) and brine (50 ml) thendried over Na₂SO₄, then filtered and evaporated to an orange oil. Thecrude material was purified by flash column chromatography on C18 (120g). The column was eluted with MeCN:water using the following gradient(% MeCN, column volumes): 10%, 2 CV; 10-100%, 20 CVs; 100%, 2 CV. Thedesired fractions were combined and concentrated in vacuo to remove mostof the MeCN. The residual aqueous mixture was extracted with EtOAc (100ml). The organic phase was separated, then dried over Na₂SO₄ andevaporated to afford the product as a bright orange oil (1.42 g, 69%).

¹H NMR (500 MHz, CDCl₃) δ 8.23 (d, J=9.4 Hz, 1H), 8.04 (s, 1H), 6.61 (d,J=2.0 Hz, 1H), 6.48 (ddd, J=9.4, 2.4, 1.2 Hz, 1H), 3.33 (qd, J=7.2, 5.1Hz, 2H), 1.39 (t, J=7.2 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=251 [MH⁺], R_(t)=1.32 min, UV purity=100%.

Intermediate 30—Synthesis of tert-butylN-{[1-ethyl-6-(trifluoromethoxy)-1H-1,3-benzodiazol-2-yl]methyl}carbamate

A suspension of palladium on carbon (10 wt %, 0.3 g) andN-ethyl-2-nitro-5-(trifluoromethoxy)aniline, Intermediate 29 (1.42 g,5.68 mmol) solution in EtOH (40 ml) was stirred under a hydrogenatmosphere at RT for 16 h. The reaction mixture was filtered throughglass fibre filter paper and washed through with EtOAc. The filtrate wasconcentrated in vacuo and then diluted with THF (10 ml). The resultingsolution was added to a pre-mixed solution of2-{[(tert-butoxy)carbonyl]amino}acetic acid (994 mg, 5.68 mmol), HATU(2.37 g, 6.24 mmol) and DIPEA (1.98 ml, 11.4 mmol) in DMF (20 ml). Thereaction mixture was stirred at RT for 68 h then poured onto saturatedaqueous NaHCO₃ (80 ml). EtOAc (50 ml) and water (50 ml) were added andthe phases were separated. The aqueous phase was extracted with EtOAc(2×50 ml) then the combined organic phases were washed with water (4×50ml) and brine (50 ml) then dried over Na₂SO₄, filtered and evaporated toa black oil. The oil thus obtained was dissolved in acetic acid (10 ml)and the resulting solution was heated at 70° C. for 1.5 h. The reactionsolution was concentrated in vacuo then the residue was partitionedbetween EtOAc (50 ml) and saturated aqueous NaHCO₃ solution (50 ml). Theorganic phase was washed with saturated aqueous NaHCO₃ solution (4×50ml), water (50 ml) and brine (10 ml) then dried over Na₂SO₄, filteredand evaporated to a brown solid (1.95 g). The crude material wasdissolved in CH₂Cl₂/MeOH then evaporated onto silica (10 g) and purifiedby flash column chromatography on a silica column (120 g). The columnwas eluted with EtOAc:heptane, increasing the gradient linearly from0:100 to 100:0 over 10 column volumes. The desired fractions werecombined and evaporated to yield the product as a pink solid (1.04 g,47%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.70-7.61 (m, 2H), 7.49 (d, J=5.1 Hz, 1H),7.19-7.10 (m, 1H), 4.44 (d, J=5.9 Hz, 2H), 4.29 (q, J=7.1 Hz, 2H),1.45-1.23 (m, 12H).

LC/MS (System A): m/z (ESI⁺)=360 [MH⁺], R_(t)=1.11 min, UV purity=92%.

Intermediate 31—Synthesis of2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-3-methyl-6-(trifluoromethoxy)-1H-1,3-benzodiazol-3-iumiodide

Iodomethane (208 μl, 3.34 mmol) was added to a suspension of tert-butylN-{[1-ethyl-6-(trifluoromethoxy)-1H-1,3-benzodiazol-2-yl]methyl}carbamate, Intermediate 30 (92%, 400 mg, 1.02 mmol) in MeCN (5 ml) in apressure tube. The tube was sealed then heated at 75° C. for 5 h thenallowed to cool to RT. The reaction mixture was concentrated in vacuo toyield the product as a dark green solid (552 mg, >99%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.30 (s, 1H), 8.18 (d, J=9.1 Hz, 1H), 7.95(t, J=5.2 Hz, 1H), 7.76 (d, J=9.1 Hz, 1H), 4.75 (d, J=5.4 Hz, 2H), 4.64(q, J=7.1 Hz, 2H), 4.10 (s, 3H), 1.44-1.25 (m 12H).

LC/MS (System A): m/z (ESI⁺)=374 [M⁺], R_(t)=0.96 min, UV purity=93%.

Intermediate 32—Synthesis of2-(aminomethyl)-1-ethyl-3-methyl-6-(trifluoromethoxy)-1H-1,3-benzodiazol-3-iumhydrochloride iodide

HCl solution in dioxane (4.0 M, 1.4 ml, 5.6 mmol) was added to asolution of2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-3-methyl-6-(trifluoromethoxy)-1H-1,3-benzodiazol-3-iumiodide, Intermediate 31 (93%, 550 mg, 1.02 mmol) in MeCN (5 ml). Thereaction was left to stir at RT for 16 h then concentrated in vacuo. Thesolid was azeotroped with MeCN (10 ml) then dried under vacuum to yieldthe product as a brown solid (480 mg, 94%).

¹H NMR (500 MHz, DMSO-d₆) δ 9.11 (s, 3H), 8.40 (d, J=1.6 Hz, 1H), 8.28(d, J=9.1 Hz, 1H), 7.83 (dd, J=9.1, 1.2 Hz, 1H), 4.80 (s, 2H), 4.75 (q,J=7.2 Hz, 2H), 4.21 (s, 3H), 1.42 (t, J=7.2 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=274 [M⁺], R_(t)=0.37 min, ELS purity=87%.

Intermediate 33—Synthesis of6-chloro-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide

A mixture of2-[(6-chloro-1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione,Intermediate 20 (850 mg, 2.50 mmol) and iodomethane (2.0 ml, 25 mmol) inMeCN (12 ml) was heated under microwave irradiation for 3 h at 120° C.The resulting solution was left to stand at RT for 64 h, resulting inprecipitation of a solid. The solid was collected by filtration thenwashed with MeCN and dried under vacuum to afford the product as a paleyellow solid (850 mg, 67%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.41 (d, J=1.8 Hz, 1H), 8.18 (d, J=8.9 Hz,1H), 7.98-7.94 (m, 2H), 7.93-7.89 (m, 2H), 7.79 (dd, J=8.9, 1.9 Hz, 1H),5.43 (s, 2H), 4.74-4.66 (m, 4H), 1.46-1.38 (m, 6H).

LC/MS (System A): m/z (ESI⁺)=368 [M(³⁵Cl)+], 370 [M(³⁷Cl)⁺], R_(t)=0.93min, UV purity=98%.

Intermediate 34—Synthesis of2-(aminomethyl)-6-chloro-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide

Hydrazine hydrate (787 μl, 16.2 mmol) was added to a suspension of6-chloro-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 33 (845 mg, 1.62 mmol) in MeOH (6 ml) in a pressuretube. The tube was sealed and heated at 80° C. for 4 h then allowed tocool to RT. The reaction mixture was concentrated in vacuo. Theresultant residue was suspended in CH₂Cl₂:MeOH (9:1, 20 ml) thenfiltered. The filtrate was concentrated in vacuo to afford the productas a yellow solid (301 mg, 49%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.32 (d, J=1.8 Hz, 1H), 8.11 (d, J=8.9 Hz,1H), 7.73 (dd, J=8.8, 1.9 Hz, 1H), 4.65-4.56 (m, 4H), 4.29 (s, 2H),1.46-1.40 (m, 6H).

LC/MS (System B): m/z (ESI⁺)=238 [M(³⁵Cl)⁺], 240 [M(³⁷Cl)⁺], R_(t)=2.00min, UV purity=97%.

Intermediate 35—Synthesis of2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-6-methoxy-1H-1,3-benzodiazol-3-iumiodide

Iodoethane (715 μl, 8.95 mmol) was added to a suspension of2-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione,Intermediate 23 (3.00 g, 8.95 mmol) in MeCN (20 ml). The mixture washeated at 80° C. for 4 h. Iodoethane (715 μl, 8.95 mmol) was added andmixture was stirred at 80° C. for 16 h. Iodoethane (715 μl, 8.95 mmol)was added and mixture was stirred at 80° C. for a further 24 h theallowed to cool to RT. The mixture was concentrated in vacuo toapproximately one third of the original volume. The precipitate wascollected by filtration then washed with MeCN to afford a grey solid(2.6 g). The filtrate was concentrated to afford a dark grey solid. Thetwo batches of solid thus obtained were combined and suspended in MeCN(20 ml). Iodoethane (715 μl, 8.95 mmol) was added then the reactionmixture was stirred at 80° C. for 18 h then at 100° C. for 4 h. Thereaction mixture was split into two equal portions in pressure tubes.Iodoethane (300 μl, 3.75 mmol) was added to both reaction mixtures thenthe pressure tubes were sealed and left to heat at 100° C. for 16 h. Thereaction mixtures were allowed to cool to RT then combined. Theresultant mixture was concentrated in vacuo to ˜5 ml then filtered. Thecollected solid was washed with the minimum of MeCN (0.5 ml) to yieldthe product as a grey solid (2.37 g). The filtrate was concentratedunder reduced pressure to afford a dark brown solid, which wastriturated with EtOAc (˜10 ml) and filtered. The filtrate was left tostand for 16 h then it was filtered again. The solids obtained from theEtOAc filtrations were combined and dried to yield an additional batchof the product as a grey solid (1.24 g). The two batches of productobtained were combined as an EtOAc suspension then evaporated and driedunder vacuum to afford the product as a grey solid (3.61 g, 81%).

¹H NMR (250 MHz, DMSO-d₆) δ 8.02 (d, J=9.2 Hz, 1H), 7.98-7.86 (m, 4H),7.63 (d, J=2.2 Hz, 1H), 7.31 (dd, J=9.1, 2.3 Hz, 1H), 5.40 (s, 2H),4.74-4.60 (m, 4H), 3.92 (s, 3H), 1.49-1.33 (m, 6H).

LC/MS (System A): m/z (ESI⁺)=364 [M⁺], R_(t)=0.93 min, UV purity=99%.

Intermediate 36—Synthesis of2-(aminomethyl)-1,3-diethyl-6-methoxy-1H-1,3-benzodiazol-3-ium iodide

Hydrazine hydrate (1.18 ml, 24.1 mmol) was added to a suspension of2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-6-methoxy-1H-1,3-benzodiazol-3-iumiodide, Intermediate 35 (2.37 g, 4.82 mmol) in MeOH (25 ml). The mixturewas then heated at 75° C. for 2.5 h then left to cool to RT over 16 h.The reaction mixture was concentrated in vacuo and the resulting solidwas suspended in CH₂Cl₂:MeOH (10:1) then filtered. The collected solidwas washed with CH₂Cl₂. The filtrate was concentrated in vacuo to affordthe product as a grey solid (1.89 g, >99%).

¹H NMR (250 MHz, DMSO-d₆) δ 7.95 (d, J=9.1 Hz, 1H), 7.58 (d, J=2.3 Hz,1H), 7.25 (dd, J=9.1, 2.3 Hz, 1H), 4.65-4.50 (m, 4H), 4.25 (s, 2H), 3.91(s, 3H), 1.50-1.39 (m, 6H).

LC/MS (System A): m/z (ESI⁺)=234 [M⁺], R_(t)=0.16 min, ELS purity=92%.

Intermediate 37—Synthesis of3-benzyl-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-iumbromide

A mixture of2-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione,Intermediate 23 (500 mg, 1.49 mmol) and (bromomethyl)benzene (531 μl,4.47 mmol) in MeCN (5 ml) was heated at 80° C. in a sealed tube for 16h. The reaction mixture allowed to cool to RT then filtered. The solidwas washed with MeCN then dried under vacuum to afford the product as awhite solid (665 mg, 87%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.84-7.79 (m, 3H), 7.78-7.74 (m, 2H), 7.71(d, J=2.3 Hz, 1H), 7.29 (dd, J=9.2, 2.3 Hz, 1H), 7.09 (t, J=7.7 Hz, 2H),7.00-6.94 (m, 3H), 5.85 (s, 2H), 5.50 (s, 2H), 4.79 (q, J=7.2 Hz, 2H),3.94 (s, 3H), 1.51 (t, J=7.2 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=426 [M⁺], R_(t)=1.02 min, UV purity=99%.

Intermediate 38—Synthesis of2-(aminomethyl)-3-benzyl-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-iumbromide

A mixture of3-benzyl-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-iumbromide, Intermediate 37 (665 mg, 1.31 mmol) and hydrazine hydrate (638μl, 13.1 mmol) in MeOH (5 ml) was stirred at 80° C. for 2 h in a sealedtube. The reaction mixture was allowed to cool then concentrated invacuo to a yellow solid. The residue was suspended in CH₂Cl₂ (20 ml)with sonication. The resultant suspension was filtered then the solidwas re-suspended in CH₂Cl₂:MeOH (1:1, 30 ml) with sonication. Theresultant suspension was filtered then the combined filtrates wereconcentrated in vacuo to afford the product as a yellow solid (526 mg,60%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.71 (d, J=9.1 Hz, 1H), 7.62 (d, J=2.3 Hz,1H), 7.41-7.31 (m, 5H), 7.20 (dd, J=9.1, 2.3 Hz, 1H), 5.86 (s, 2H), 4.63(q, J=7.2 Hz, 2H), 4.34 (s, 2H), 3.90 (s, 3H), 1.48 (t, J=7.2 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=296 [M⁺], R_(t)=0.76 min, UV purity=56%.

Intermediate 39—Synthesis of tert-butylN-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]carbamate

HATU (8.53 g, 22.3 mmol) and DIPEA (7.1 ml, 41 mmol) were added to asolution of N-(tert-butoxycarbonyl)glycine (3.93 g, 22.4 mmol) in DMF(40 ml). The resulting solution was stirred at RT for 0.5 h then asolution of 1-N-ethyl-5-methoxybenzene-1,2-diamine (3.39 g, 20.4 mmol)in THF (20 ml) was added. The reaction was left to stir at RT for 80 minthen the reaction mixture was diluted with EtOAc (200 ml) and water (150ml). The phases were separated then the organic phase was washed withwater (3×150 ml) and brine (150 ml). The combined organic phases weredried over Na₂SO₄ then concentrated in vacuo to afford the crudeintermediate as a dark red viscous oil. The intermediate was taken up inAcOH (40 ml) and the resulting solution was stirred at 60° C. for 18 h.The reaction mixture was allowed to cool then concentrated in vacuo. Theresidue was then dissolved in EtOAc (200 ml) then the pH was adjusted to9 by the addition of saturated aqueous NaHCO₃ solution. The phases wereseparated and the organic phase was washed with water (2×150 ml) andbrine (150 ml), then dried over Na₂SO₄ and concentrated in vacuo toafford the crude product as a dark red oil. The crude material waspurified by flash column chromatography on a silica column (340 g). Thecolumn was eluted with CH₂Cl₂:MeOH, increasing the gradient linearlyfrom 100:0 to 92:8 over 10 column volumes. The desired fractions werecombined and evaporated to a viscous dark red oil which solidified onstanding to yield the product as a dark red solid (5.02 g, 77%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.47-7.39 (m, 2H), 7.06 (d, J=2.4 Hz, 1H),6.78 (dd, J=8.7, 2.4 Hz, 1H), 4.39 (d, J=5.9 Hz, 2H), 4.22 (q, J=7.1 Hz,2H), 3.81 (s, 3H), 1.39 (s, 9H), 1.28 (t, J=7.2 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=306 [MH⁺], R_(t)=0.88 min, UV purity=96%.

Intermediate 40—Synthesis of2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-6-methoxy-3-(2-methoxy-2-oxoethyl)-1H-1,3-benzodiazol-3-iumbromide

Methyl bromoacetate (395 μl, 4.18 mmol) was added to a suspension oftert-butylN-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]carbamate,Intermediate 39 (85%, 500 mg, 1.39 mmol) in MeCN (4 ml) in a pressuretube. The tube was sealed and the resulting mixture was stirred at 75°C. for 4 h then allowed to cool to RT. The resultant suspension wasfiltered then the solid was washed with cold MeCN and dried under vacuumto afford the product as a white solid (454 mg, 71%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.90 (d, J=9.2 Hz, 1H), 7.86 (t, J=5.3 Hz,1H), 7.62 (d, J=2.2 Hz, 1H), 7.29 (dd, J=9.1, 2.3 Hz, 1H), 5.58 (s, 2H),4.75 (d, J=5.4 Hz, 2H), 4.65 (q, J=7.2 Hz, 2H), 3.92 (s, 3H), 3.75 (s,3H), 1.43 (t, J=7.2 Hz, 3H), 1.35 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=378 [M⁺], R_(t)=0.88 min, UV purity=100%.

Intermediate 41—Synthesis of2-(aminomethyl)-1-ethyl-6-methoxy-3-(2-methoxy-2-oxoethyl)-1H-1,3-benzodiazol-3-iumhydrochloride bromide

A mixture of2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-6-methoxy-3-(2-methoxy-2-oxoethyl)-1H-1,3-benzodiazol-3-iumbromide, Intermediate 40 (454 mg, 0.990 mmol) and HCl solution indioxane (4.0 M, 2.5 ml, 10 mmol) was stirred at RT for 0.5 h. Thereaction mixture was concentrated in vacuo to afford the product as apale orange foam (580 mg, 96%—yield corrected for 35 wt % residualdioxane observed in NMR).

¹H NMR (500 MHz, DMSO-d₆)¹H NMR (500 MHz, DMSO-d₆) δ 8.86 (s, 3H), 7.97(d, J=9.2 Hz, 1H), 7.68 (d, J=2.3 Hz, 1H), 7.35 (dd, J=9.2, 2.3 Hz, 1H),5.77 (s, 2H), 4.79 (s, 2H), 4.72 (q, J=7.2 Hz, 2H), 3.94 (s, 3H), 3.77(s, 3H), 1.47 (t, J=7.2 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=278 [M⁺], R_(t)=0.14 min, ELS purity=95%.

Intermediate 42—Synthesis of2-({[(tert-butoxy)carbonyl]amino}methyl)-3-(carbamoylmethyl)-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-iumbromide

2-Bromoacetamide (691 mg, 5.01 mmol) was added to a suspension oftert-butylN-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]carbamate,Intermediate 39 (85%, 600 mg, 1.67 mmol) in MeCN (6 ml) in a pressuretube. The tube was sealed then the reaction mixture was stirred at 80°C. for 5 h. The reaction was allowed to cool to RT then stirred at RTfor 64 h. The reaction mixture was heated to 80° C. for a further 2 hthen allowed to cool to RT. The resultant mixture was concentrated invacuo to a viscous red oil. The crude material was purified by flashcolumn chromatography on a silica column (50 g). The column was elutedwith CH₂Cl₂:MeOH, using the following gradient (% MeOH, column volumes):0%, 1 CV; 0-9%, 8 CV; 9%, 3 CV; 9-14%, 2 CV, 14%, 1 CV; 14-20%, 2 CV.The desired fractions were combined and evaporated to afford the productas a pale magenta foam (638 mg, 84%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.90 (s, 1H), 7.86-7.77 (m, 2H), 7.63 (s,1H), 7.60 (d, J=2.2 Hz, 1H), 7.29 (dd, J=9.1, 2.3 Hz, 1H), 5.30 (s, 2H),4.70 (d, J=5.4 Hz, 2H), 4.62 (q, J=7.2 Hz, 2H), 3.92 (s, 3H), 1.42 (t,J=7.2 Hz, 3H), 1.36 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=363 [M⁺], R_(t)=0.83 min, UV purity=98%.

Intermediate 43—Synthesis of2-(aminomethyl)-3-(carbamoylmethyl)-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-iumhydrochloride bromide

A mixture of2-({[(tert-butoxy)carbonyl]amino}methyl)-3-(carbamoylmethyl)-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-iumbromide, Intermediate 42 (98%, 638 mg, 1.41 mmol) and HCl solution indioxane (4.0 M, 3.5 ml, 14 mmol) was stirred at RT for 0.5 h. Thereaction mixture was concentrated in vacuo to afford the product as apurple solid (719 mg, 97%—yield corrected for 28 wt % residual dioxaneobserved in NMR).

¹H NMR (500 MHz, DMSO-d₆) δ 8.91 (s, 3H), 8.35 (s, 1H), 7.98-7.89 (m,2H), 7.68 (d, J=2.3 Hz, 1H), 7.38 (dd, J=9.2, 2.3 Hz, 1H), 5.45 (s, 2H),4.77 (s, 2H), 4.71 (q, J=7.2 Hz, 2H), 3.94 (s, 3H), 1.46 (t, J=7.2 Hz,3H).

LC/MS (System A): m/z (ESI⁺)=263 [M⁺], R_(t)=0.15 min, ELS purity=100%.

Intermediate 44—Synthesis of2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-6-methoxy-3-[2-(methylsulfanyl)ethyl]-1H-1,3-benzodiazol-3-iumiodide

A mixture of2-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione,Intermediate 23 (500 mg, 1.49 mmol), 1-chloro-2-(methylsulfanyl)ethane(730 μl, 7.45 mmol) and NaI (1.12 g, 7.45 mmol) in MeCN (8 ml) washeated to 100° C. in a sealed tube for 5 h then allowed to cool to RT.The reaction mixture was concentrated in vacuo to a brown solid. Thesolid thus obtained was suspended in MeOH (8 ml) with sonication. Theresultant suspension was filtered then the solid was washed with MeOHand dried under vacuum to afford the product as a brown solid (1.13 g,50%)

¹H NMR (500 MHz, DMSO-d₆) δ 8.04 (d, J=9.2 Hz, 1H), 7.99-7.88 (m, 4H),7.64 (t, J=2.6 Hz, 1H), 7.32 (dd, J=9.2, 2.3 Hz, 1H), 5.47-5.37 (m, 2H),4.90-4.68 (m, 4H), 4.11 (s, 1H), 3.92 (d, J=1.4 Hz, 3H), 3.03 (t, J=7.1Hz, 2H), 2.15 (s, 2H), 1.42 (q, J=6.9 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=410 [M⁺], R_(t)=0.99 min, UV purity=66%.

Intermediate 45—Synthesis of2-(aminomethyl)-1-ethyl-6-methoxy-3-[2-(methylsulfanyl)ethyl]-1H-1,3-benzodiazol-3-iumiodide

A mixture of2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-6-methoxy-3-[2-(methylsulfanyl)ethyl]-1H-1,3-benzodiazol-3-iumiodide, Intermediate 44 (66%, 1.00 g, 1.62 mmol) and hydrazine hydrate(226 μl, 4.65 mmol) in MeOH (5 ml) was heated to 80° C. in a sealed tubefor 80 min. The reaction mixture was concentrated in vacuo to an orangesolid. The solid thus obtained was suspended in CH₂Cl₂ (50 ml) withsonication. The suspension was filtered then the filtrate wasconcentrated in vacuo to afford the product as a pale orange solid (395mg, 48%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.96 (d, J=9.1 Hz, 1H), 7.60-7.56 (m, 1H),7.27 (dt, J=9.1, 2.1 Hz, 1H), 4.76 (t, J=6.9 Hz, 2H), 4.61 (q, J=6.1,4.9 Hz, 2H), 4.30 (s, 2H), 3.92 (d, J=1.4 Hz, 3H), 3.01 (t, J=6.9 Hz,2H), 2.41-2.30 (m, 2H), 2.13 (s, 3H), 1.45 (t, J=7.3 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=280 [M⁺], R_(t)=0.65 min, UV purity=80%.

Intermediate 46—Synthesis of2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-3-(2-hydroxyethyl)-6-methoxy-1H-1,3-benzodiazol-3-iumbromide

(2-Bromoethoxy)(tert-butyl)dimethylsilane (1.66 g, 6.96 mmol) was addedto a suspension of tert-butylN-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]carbamate,Intermediate 39 (85%, 500 mg, 1.39 mmol) in MeCN (5 ml) in a pressuretube. The tube was sealed then the reaction mixture was stirred at 80°C. for 0.5 h then at 100° C. for 16 h. The reaction was allowed to coolto RT then additional (2-bromoethoxy)(tert-butyl)dimethylsilane (1.00 g,4.18 mmol) was added. The tube was sealed then the reaction was left tostir at 100° C. for a further 24 h. The reaction mixture was allowed tocool then concentrated in vacuo to a red oil. The crude material waspurified by flash column chromatography on a silica column (25 g). Thecolumn was eluted with CH₂Cl₂:MeOH, increasing the gradient linearlyfrom 100:0 to 90:10 over 10 column volumes. The desired fractions werecombined and evaporated to yield a viscous dark red oil (615 mg). Thematerial was further purified by flash column chromatography on C18 (30g). The column was eluted with MeCN:water+0.1% formic acid using thefollowing gradient (% MeCN, column volumes): 10%, 2 CV; 10-36%; 36%, 2CV; 36-50%, 4 CV; 50-100%, 3 CV; 100%, 3 CV. The desired fractions werecombined and evaporated to yield the product as a viscous dark red oil(278 mg, 43%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.28 (s, 1H), 7.99-7.84 (m, 2H), 7.57 (d,J=2.2 Hz, 1H), 7.28 (dd, J=9.1, 2.3 Hz, 1H), 4.76 (d, J=5.5 Hz, 2H),4.71-4.65 (m, 2H), 4.59 (q, J=7.1 Hz, 2H), 3.92 (s, 3H), 3.77 (t, J=4.7Hz, 2H), 1.41 (t, J=7.2 Hz, 3H), 1.38 (d, J=2.8 Hz, 9H).

LC/MS (System A): m/z (ESI⁺)=350 [MH⁺], R_(t)=0.88 min, UV purity=92%.

Intermediate 47—Synthesis of2-(aminomethyl)-1-ethyl-3-(2-hydroxyethyl)-6-methoxy-1H-1,3-benzodiazol-3-iumhydrochloride bromide

Hydrogen chloride solution in dioxane (4.0 M, 1.2 ml, 4.8 mmol) wasadded to a solution of2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-3-(2-hydroxyethyl)-6-methoxy-1H-1,3-benzodiazol-3-iumbromide, Intermediate 46 (92%, 227 mg, 0.49 mmol) in MeCN (3 ml). Theresulting mixture was stirred at RT for 1 h. The reaction mixture wasconcentrated in vacuo to afford the product as a dark purple solid (174mg, 97%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.89 (s, 3H), 8.03 (d, J=9.2 Hz, 1H), 7.66(d, J=2.3 Hz, 1H), 7.35 (dd, J=9.2, 2.3 Hz, 1H), 4.82-4.73 (m, 4H), 4.69(q, J=7.2 Hz, 2H), 3.94 (s, 3H), 3.80 (t, J=4.6 Hz, 2H), 1.45 (t, J=7.2Hz, 3H).

LC/MS (System B): m/z (ESI⁺)=250 [MH⁺], R_(t)=1.58 min, UV purity=99%.

Intermediate 48—Synthesis of2-{[(2-carboxyphenyl)formamido]methyl}-1-ethyl-3-{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}-6-methoxy-1H-1,3-benzodiazol-3-iumiodide

A mixture of2-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione,Intermediate 23 (2.00 g, 5.96 mmol) and2-[2-(2-iodoethoxy)ethoxy]ethan-1-ol (90%, 8.62 g, 29.8 mmol) in MeCN(15 ml) was heated at 130° C. in a sealed tube for 16 h then allowed tocool to RT. The reaction mixture was concentrated in vacuo then thecrude material was purified by flash column chromatography on C18 (12g). The column was eluted with MeCN:water+0.1% formic acid using thefollowing gradient (% MeCN, column volumes): 10%, 2 CV; 10-14%, 2 CV;14-18%, 1 CV; 18-27%, 2 CV; 27-31%, 0.5 CV; 31-60%, 0.5 CV; 60-100%, 1CV; 100%, 1 CV. The desired fractions were combined and evaporated toyield the product as a yellow amorphous solid (1.72 g, 47%).

¹H NMR (500 MHz, DMSO-d₆) δ 12.97-12.91 (m, 1H), 7.96 (d, J=9.2 Hz, 1H),7.72 (dd, J=7.8, 1.2 Hz, 1H), 7.60 (dd, J=7.7, 1.3 Hz, 1H), 7.57 (d,J=2.3 Hz, 1H), 7.37 (td, J=7.5, 1.4 Hz, 1H), 7.30-7.24 (m, 2H), 5.04 (d,J=5.0 Hz, 2H), 4.91 (t, J=4.8 Hz, 2H), 4.77-4.62 (m, 3H), 3.91 (s, 3H),3.83 (t, J=4.9 Hz, 2H), 3.51-3.46 (m, 2H), 3.43-3.39 (m, 4H), 3.36-3.28(m, 2H+HDO), 1.41 (t, J=7.2 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=486 [M⁺], R_(t)=0.80 min, UV purity=100%.

Intermediate 49—Synthesis of2-(aminomethyl)-1-ethyl-3-{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}-6-methoxy-1H-1,3-benzodiazol-3-iumiodide

Hydrazine hydrate (639 μl, 13.2 mmol) was added to a solution of2-{[(2-carboxyphenyl)formamido]methyl}-1-ethyl-3-{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}-6-methoxy-1H-1,3-benzodiazol-3-iumiodide, Intermediate 48 (1.52 g, 2.48 mmol) in MeOH (12 ml) in apressure tube. The tube was sealed and the reaction solution was heatedat 75° C. for 3.5 h. The reaction was allowed to cool to RT then stirredat RT for 64 h. Additional hydrazine hydrate (639 μl, 13.2 mmol) wasadded and the reaction was heated at 75° C. for a further 18 h, then at80° C. for a further 24 h. The reaction mixture was allowed to cool toRT then concentrated in vacuo to an orange solid. The solid thusobtained was suspended in CH₂Cl₂:MeOH (9:1, 30 ml) then filtered. Thefiltrate was concentrated in vacuo to afford the product as a viscousorange oil (1.21 g, 88%).

LC/MS (System B): m/z (ESI⁺)=338 [M⁺], Rt=1.41 min, UV purity=84%.

Intermediate 50—Synthesis of3-benzyl-6-chloro-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-3-iumbromide

Benzyl bromide (88 μl, 0.74 mmol) was added to a suspension of2-[(6-chloro-1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione,Intermediate 20 (0.25 g, 0.74 mmol) in MeCN (5 ml) in a pressure tube.The tube was sealed then the reaction was stirred at 80° C. for 16 hthen allowed to cool to RT. Benzyl bromide (88 μl, 0.74 mmol) was addedthen the reaction was stirred at 80° C. for a further 6 h. The reactionwas allowed to cool to RT then left to stand at RT for 64 h. Benzylbromide (88 μl, 0.74 mmol) was added then the reaction was stirred at80° C. for a further 24 h. The reaction was allowed to cool to RT thenfiltered. The solid was washed with MeCN then dried under vacuum toafford the product as a white solid (370 mg, 97%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.50 (d, J=1.8 Hz, 1H), 7.98 (d, J=8.9 Hz,1H), 7.85-7.68 (m, 5H), 7.14-7.05 (m, 2H), 6.99 (t, J=6.5 Hz, 3H), 5.89(s, 2H), 5.52 (s, 2H), 4.82 (q, J=7.2 Hz, 2H), 1.51 (t, J=7.2 Hz, 3H).

LC/MS (System B): m/z (ESI⁺)=430 [M⁺], Rt=0.98 min, UV purity=99%.

Intermediate 51—Synthesis of2-(aminomethyl)-3-benzyl-6-chloro-1-ethyl-1H-1,3-benzodiazol-3-iumbromide

Hydrazine hydrate (0.18 ml, 3.62 mmol) was added to a suspension of3-benzyl-6-chloro-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-3-iumbromide, Intermediate 50 (370 mg, 0.724 mmol) in MeOH (4 ml) in apressure tube. The tube was sealed then the reaction was stirred at 75°C. for 3 h. The reaction mixture was allowed to cool to RT thenfiltered. The filtrate was concentrated in vacuo to afford an orangesolid. The solid thus obtained was suspended in CH₂Cl₂:MeOH (9:1, 10 ml)then filtered. The solid was dried under vacuum then suspended in MeOH.The suspension was filtered then the filtrate was concentrated in vacuo.The resultant residue was suspended in CH₂Cl₂:MeOH (9:1, 10 ml) withsonication. The resultant suspension was filtered then the filtrate wasconcentrated in vacuo to afford the product as a pale yellow solid (107mg, 33%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.36 (d, J=1.8 Hz, 1H), 7.83 (d, J=8.9 Hz,1H), 7.66 (dd, J=8.9, 1.9 Hz, 1H), 7.43-7.30 (m, 5H), 5.89 (s, 2H), 4.63(q, J=7.2 Hz, 2H), 4.35 (s, 2H), 1.46 (t, J=7.2 Hz, 3H).

LC/MS (System B): m/z (ESI⁺)=300 [M⁺], Rt=0.81 min, UV purity=85%.

Intermediate 52—Synthesis of3-benzyl-2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-iumbromide

Benzyl bromide (173 μl, 1.46 mmol) was added to a suspension oftert-butylN-{[1-ethyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-2-yl]methyl}carbamate,Intermediate 26 (250 mg, 0.73 mmol) in MeCN (3 ml) in a pressure tube.The tube was sealed and heated at 80° C. for 16 h then the reactionmixture was concentrated in vacuo to afford an orange oil whichsolidified on standing. The resulting solid was suspended in MeCN (2ml). The solid was collected by filtration then dried under vacuum toafford the product as a white solid (160 mg). The filtrate wasconcentrated in vacuo. The residue was suspended in the minimum volumeof MeCN then filtered. The solid thus obtained was dried under vacuum toyield a second batch of product as an off-white solid (90 mg). The 2batches of product were combined in MeCN then evaporated to afford theproduct as an off-white solid (250 mg, 64%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.69 (s, 1H), 8.03-7.87 (m, 3H), 7.43-7.36(m, 3H), 7.31-7.23 (m, 2H), 5.92 (s, 2H), 4.97-4.85 (m, 2H), 4.78 (q,J=7.1 Hz, 2H), 1.49 (t, J=7.1 Hz, 3H), 1.30 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=435 [M⁺], R_(t)=1.05 min, UV purity=96%.

Intermediate 53—Synthesis of2-(aminomethyl)-3-benzyl-1-ethyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-iumhydrochloride bromide

HCl solution in dioxane (4.0 M, 0.61 ml, 2.4 mmol) was added to asolution of3-benzyl-2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-iumbromide, Intermediate 52 (250 mg, 0.49 mmol) in MeCN (2.5 ml). Thereaction was stirred at RT for 18 h then concentrated in vacuo. Theresidue was azeotroped with MeCN then dried under vacuum to afford theproduct as a pale yellow solid (209 mg, 95%).

¹H NMR (500 MHz, DMSO-d₆) δ 9.18 (s, 3H), 8.77 (s, 1H), 8.10-7.99 (m,2H), 7.51-7.46 (m, 2H), 7.44-7.34 (m, 3H), 6.05 (s, 2H), 4.94-4.79 (m,4H), 1.53 (t, J=7.2 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=334 [M⁺], R_(t)=0.86 min, UV purity=100%.

Intermediate 54—Synthesis of tert-butylN-[(1-benzyl-1H-1,3-benzodiazol-2-yl)methyl]carbamate

Benzyl bromide (1.29 ml, 10.8 mmol) was added to a mixture of tert-butylN-(1H-1,3-benzodiazol-2-ylmethyl)carbamate (85%, 3.15 g, 10.8 mmol) andK₂CO₃ (2.25 g, 16.3 mmol) in DMF (25 ml). The resulting mixture wasstirred at RT for 16 h. The reaction mixture was partitioned betweenwater (150 ml) and EtOAc (150 ml). The phases were separated then theaqueous phase was extracted with EtOAc (5×150 ml). The combined organicswere dried over Na₂SO₄ then concentrated in vacuo to approximately 50ml. The resultant slurry was recrystallized from the minimum volume ofrefluxing EtOAc to afford the product as white solid (2.22 g, 61%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.64-7.58 (m, 1H), 7.51-7.45 (m, 1H),7.40-7.34 (m, 1H), 7.34-7.24 (m, 3H), 7.20-7.11 (m, 4H), 5.51 (s, 2H),4.44 (d, J=5.8 Hz, 2H), 1.32 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=338 [MH⁺], R_(t)=1.02 min, UV purity=100%.

Intermediate 55—Synthesis of1-benzyl-2-({[(tert-butoxy)carbonyl]amino}methyl)-3-(2-methoxy-2-oxoethyl)-1H-1,3-benzodiazol-3-iumbromide

A mixture of tert-butylN-[(1-benzyl-1H-1,3-benzodiazol-2-yl)methyl]carbamate, Intermediate 54(500 mg, 1.48 mmol) and methyl bromoacetate (421 μl, 4.45 mmol) in MeCN(5 ml) was stirred at 70° C. in a sealed tube for 16 h. The reactionmixture was allowed to cool then concentrated in vacuo to afford theproduct as a white solid (717 mg, 96%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.06 (d, J=8.3 Hz, 1H), 7.89 (t, J=5.4 Hz,1H), 7.81 (d, J=8.2 Hz, 1H), 7.73-7.60 (m, 2H), 7.42-7.32 (m, 3H), 7.28(d, J=6.8 Hz, 2H), 5.97 (s, 2H), 5.69 (s, 2H), 4.89 (d, J=5.4 Hz, 2H),3.77 (s, 3H), 1.32 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=410 [M⁺], R_(t)=0.94 min, UV purity=97%.

Intermediate 56—Synthesis of1-benzyl-2-({[(tert-butoxy)carbonyl]amino}methyl)-3-[2-oxo-2-(piperidin-1-yl)ethyl]-1H-1,3-benzodiazol-3-iumbromide

A mixture of1-benzyl-2-({[(tert-butoxy)carbonyl]amino}methyl)-3-(2-methoxy-2-oxoethyl)-1H-1,3-benzodiazol-3-iumbromide, Intermediate 55 (350 mg, 0.71 mmol) in aqueous LiOH solution(1.0 M, 1.4 ml, 1.4 mmol) and MeOH (1.5 ml) was stirred at RT for 16 h.The reaction mixture was acidified to pH 3 by dropwise addition ofaqueous HCl solution (1 M) then concentrated in vacuo to afford thecrude intermediate as a beige solid (469 mg). The crude intermediatethus obtained was dissolved in DMF (4 ml) then HATU (340 mg, 0.89 mmol)and DIPEA (206 μl, 1.18 mmol) were added. Piperidine (88 μl, 0.89 mmol)was added to the reaction solution then the resulting mixture wasstirred at RT for 45 min. Additional piperidine (150 μl, 1.52 mmol) andHATU (300 mg, 0.89 mmol) were added and the reaction was left to stir atRT for an additional 64 h. The reaction mixture was concentrated invacuo then the crude material was purified by flash columnchromatography on C18 (30 g). The column was eluted with MeCN:water+0.1%formic acid using the following gradient (% MeCN, column volumes):10-45%, 12 CV; 45%, 2 CV; 45-53%, 3 CV; 53-100%, 3 CV; 100%, 1 CV. Thedesired fractions were combined and evaporated to afford the product asa pale orange foam (209 mg, 53%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.98 (d, J=8.3 Hz, 1H), 7.83-7.76 (m, 2H),7.70-7.58 (m, 2H), 7.43-7.32 (m, 3H), 7.25 (d, J=6.8 Hz, 2H), 5.97 (s,2H), 5.76 (s, 2H), 4.78 (d, J=5.4 Hz, 2H), 3.58-3.51 (m, 2H), 3.47-3.42(m, 2H), 1.76-1.62 (m, 4H), 1.51 (s (br), 2H), 1.32 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=463 [M⁺], R_(t)=1.00 min, UV purity=99%.

Intermediate 57—Synthesis of2-(aminomethyl)-1-benzyl-3-[2-oxo-2-(piperidin-1-yl)ethyl]-1H-1,3-benzodiazol-3-iumhydrochloride bromide

A mixture of1-benzyl-2-({[(tert-butoxy)carbonyl]amino}methyl)-3-[2-oxo-2-(piperidin-1-yl)ethyl]-1H-1,3-benzodiazol-3-iumbromide, Intermediate 56 (205 mg, 0.377 mmol) and HCl solution indioxane (4.0 M, 1.1 ml, 4.4 mmol) was stirred at RT for 0.5 h. Thereaction mixture was concentrated in vacuo to afford the product as aviscous orange oil (219 mg, 92%—yield corrected for 24 wt % residualdioxane observed in NMR).

¹H NMR (500 MHz, DMSO-d₆) δ 8.15-8.09 (m, 1H), 7.91 (d, J=8.3 Hz, 1H),7.74 (t, J=7.7 Hz, 1H), 7.68 (t, J=7.8 Hz, 1H), 7.44-7.33 (m, 3H), 7.32(d, J=7.0 Hz, 2H), 6.05 (s, 2H), 5.97 (s, 2H), 4.74 (s, 2H), 3.63-3.58(m, 2H), 3.48-3.45 (m, 2H), 1.79-1.71 (m, 2H), 1.71-1.63 (m, 2H),1.57-1.46 (m, 2H).

LC/MS (System A): m/z (ESI⁺)=363 [M⁺], R_(t)=0.82 min, UV purity=93%.

Intermediate 58—Synthesis of6-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)-1,3-diethyl-2-({[(9H-fluoren-9-ylmethoxy)carbonyl]amino}methyl)-1H-1,3-benzodiazol-3-iumiodide

Intermediate 58 was synthesised by according to literature procedures(US 2015/0018313 A1).

Intermediate 59—Synthesis of2-(aminomethyl)-6-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide

Morpholine (3.46 ml, 40.0 mmol) was added to a solution of6-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)-1,3-diethyl-2-({[(9H-fluoren-9-ylmethoxy)carbonyl]amino}methyl)-1H-1,3-benzodiazol-3-iumiodide, Intermediate 58 (95%, 3.00 g, 4.00 mmol) in THF (50 ml). Thereaction mixture was stirred at RT for 25 min then diluted with diethylether (150 ml). The resulting mixture was agitated then the supernatantwas decanted off. The residual gum was washed further with ether (2×60ml) then dried under vacuum. The residue was dissolved in THF thenconcentrated in vacuo afford the product as a pale orange solid (1.99 g,83%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.95 (d, J=9.1 Hz, 1H), 7.65-7.61 (m, 1H),7.25 (dd, J=9.1, 2.3 Hz, 1H), 7.11-7.05 (m, 1H), 4.61-4.53 (m, 4H), 4.25(s, 2H), 4.11 (t, J=5.9 Hz, 2H), 3.36 (q, J=5.9 Hz, 2H), 1.45-1.41 (m,6H), 1.39 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=363 [M⁺], R_(t)=0.76 min, UV purity=82%.

Intermediate 60—Synthesis tert-butylN-[3-(3-fluoro-4-nitrophenoxy)propyl]carbamate

A suspension of 3-fluoro-4-nitrophenol (2.50 g, 15.9 mmol), tert-butyl(3-bromopropyl)carbamate (3.98 g, 16.7 mmol) and K₂CO₃ (2.64 g, 19.1mmol) in acetone (15 ml) was stirred at 60° C. for 18 h. The reactionmixture was concentrated in vacuo then the residue was partitionedbetween EtOAc (50 ml) and water (50 ml). The phases were separated thenthe organic phase was extracted with water (2×50 ml) and brine (50 ml)then dried over Na₂SO₄ and concentrated in vacuo to afford the productas a viscous orange oil (4.65 g, 84%).

¹H NMR (500 MHz, Acetone-d6) δ 8.14 (t, J=9.2 Hz, 1H), 7.16 (dd, J=13.7,2.5 Hz, 1H), 6.96 (dd, J=9.3, 2.6 Hz, 1H), 6.92 (t, J=5.5 Hz, 1H), 4.13(t, J=6.2 Hz, 2H), 3.07 (q, J=6.6 Hz, 2H), 1.84 (p, J=6.5 Hz, 2H), 1.37(s, 9H).

LC/MS (System A): R_(t)=1.22 min, UV purity=90%.

Intermediate 61—Synthesis of tert-butylN-{3-[3-(ethylamino)-4-nitrophenoxy]propyl}carbamate

Ethylamine solution in THF (2.0 M, 10 ml, 20 mmol) was added to amixture of tert-butyl N-[3-(3-fluoro-4-nitrophenoxy)propyl]carbamate,Intermediate 60 (90%, 4.65 g, 13.3 mmol) and K₂CO₃ (2.20 g, 16.0 mmol)in THF (30 ml). The reaction mixture was stirred at RT for 16 h thenadditional ethylamine solution in THF (2.0 M, 3.0 ml, 6.0 mmol) wasadded. The reaction mixture was left to stir at RT for a further 70 hthen filtered. The filter pad was rinsed with EtOAc then the combinedfiltrate was extracted with water (2×150 ml) and brine (150 ml) thendried over Na₂SO₄ and concentrated in vacuo to afford the product as aviscous yellow/orange oil (4.69 g, 93%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.26 (t, J=5.0 Hz, 1H), 8.02 (d, J=10.0 Hz,1H), 6.92-6.86 (m, 1H), 6.30-6.27 (m, 2H), 4.09 (t, J=6.3 Hz, 2H),3.40-3.35 (m, 2H), 3.08 (q, J=6.7 Hz, 2H), 1.84 (p, J=6.5 Hz, 2H), 1.37(s, 9H), 1.24 (t, J=7.1 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=340 [MH⁺], R_(t)=1.30 min, UV purity=90%.

Intermediate 62—Synthesis of (9H-fluoren-9-yl)methylN-{[6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1-ethyl-1H-1,3-benzodiazol-2-yl]methyl}carbamate

Palladium on carbon (10 wt %, 662 mg) was added to a solution oftert-butyl N-{3-[3-(ethylamino)-4-nitrophenoxy]propyl}carbamate,Intermediate 61 (90%, 4.69 g, 12.4 mmol) in EtOH (60 ml). The resultingmixture was stirred under a hydrogen atmosphere for 20 h. The reactionmixture was filtered through a Celite pad then the filtrate wasconcentrated in vacuo. The residue was dissolved in DMF (10 ml) to givea solution of the phenylenediamine intermediate. A solution ofFMOC-glycine (3.88 g, 13.1 mmol), HATU (5.20 g, 13.7 mmol) and DIPEA(4.3 ml, 25 mmol) in DMF (20 ml) was stirred at RT for 0.5 h. Thephenylenediamine DMF solution was then added and the resulting solutionwas stirred at RT for 1 h. Additional FMOC-glycine (2.00 g, 6.73 mmoland HATU (2.50 g, 6.57 mmol) were added then the reaction was left tostir at RT for a further 45 min. The reaction mixture was partitionedbetween EtOAc (100 ml) and water (100 ml). The phases were separatedthen the organic phase was washed with water (2×100 ml) and brine (100ml) then dried over Na₂SO₄ and concentrated in vacuo to afford a redsolid. The solid thus obtained was dissolved in AcOH (20 ml) then heatedat 60° C. for 16 h. The reaction was allowed to cool to RT thenconcentrated in vacuo. The residue thus obtained was treated withsaturated aqueous NaHCO₃ solution until pH 9 then partitioned betweenEtOAc (250 ml) and water (250 ml). The phases were separated then theorganic phase was dried over Na₂SO₄ and concentrated in vacuo.

The crude material was purified by flash column chromatography on asilica column (340 g). The column was eluted with CH₂Cl₂:MeOH, using thefollowing gradient (% MeOH, column volumes): 0%, 1 CV; 0-3.3%, 7 CV;3.3%, 1 CV; 3.3-4.5%, 2 CV. The desired fractions were combined andevaporated to afford the product as a pale orange solid (4.73 g, 53%).

LC/MS (System A): m/z (ESI⁺)=571 [MH⁺], R_(t)=1.18 min, UV purity=80%.

Intermediate 63—Synthesis of6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-iumiodide

A mixture of 9H-fluoren-9-ylmethylN-{[6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1-ethyl-1H-1,3-benzodiazol-2-yl]methyl}carbamate,Intermediate 62 (80%, 1.50 g, 2.10 mmol) and iodoethane (1.69 ml, 21.0mmol) in THF (15 ml) was heated under microwave irradiation for 1.5 h at120° C. The reaction mixture was concentrated in vacuo then the crudematerial was purified by flash column chromatography on a silica column(100 g). The column was eluted with CH₂Cl₂:MeOH, using the followinggradient (% MeOH, column volumes): 0%, 1 CV; 0-3.4%, 7 CV; 3.4-4.3%, 2CV, 4.3-6.0%, 2 CV. The desired fractions were combined and evaporatedto afford the product as a pale orange foam (1.03 g, 61%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.24 (t, J=5.1 Hz, 1H), 7.95 (d, J=9.1 Hz,1H), 7.86 (d, J=7.5 Hz, 2H), 7.62 (d, J=7.5 Hz, 2H), 7.56 (s, 1H), 7.36(t, J=7.4 Hz, 2H), 7.30-7.22 (m, 3H), 6.93-6.88 (m, 1H), 4.74 (d, J=5.1Hz, 2H), 4.55-4.47 (m, 6H), 4.22 (t, J=5.8 Hz, 1H), 4.13 (t, J=6.0 Hz,2H), 3.12 (q, J=6.4 Hz, 2H), 1.94-1.85 (m, 2H), 1.37-1.31 (m, 15H).

LC/MS (System A): m/z (ESI⁺)=599 [M⁺], R_(t)=1.15 min, UV purity=90%.

Intermediate 64—Synthesis of2-(aminomethyl)-6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide

Morpholine (1.10 ml, 12.7 mmol) was added to a solution of6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1,3-diethyl-2-({[(9H-fluoren-9-ylmethoxy)carbonyl]amino}methyl)-1H-1,3-benzodiazol-3-iumiodide, Intermediate 63 (90%, 1.03 g, 1.27 mmol) in THF (10 ml). Thereaction mixture was stirred at RT for 2 h then diluted with diethylether (40 ml). The mixture was agitated then the supernatant wasdecanted off. The procedure was repeated with further diethyl etherwashes (2×20 ml). The residue thus obtained was dried under vacuum toafford the product as a pale orange foam (700 mg, 99%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.94 (d, J=9.1 Hz, 1H), 7.56 (d, J=2.1 Hz,1H), 7.25 (dd, J=9.1, 2.2 Hz, 1H), 6.99-6.83 (m, 1H), 4.56 (q, J=7.2 Hz,4H), 4.24 (s, 2H), 4.12 (t, J=6.2 Hz, 2H), 3.12 (q, J=6.6 Hz, 2H),1.94-1.84 (m, 2H), 1.42 (t, J=7.2 Hz, 6H), 1.37 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=377 [M⁺], R_(t)=0.82 min, UV purity=91%.

Intermediate 65—Synthesis of2-[(6-bromo-1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione

A mixture of N-phthaloylglycine (13.1 g, 63.8 mmol), TBTU (21.5 g, 67.0mmol) and triethylamine (14.1 ml, 79.1 mmol) in DMF (150 ml) was stirredat RT for 45 min. A solution of 5-bromo-1-N-ethylbenzene-1,2-diamine(13.1 g, 60.9 mmol) in THF (50 ml) was added and the resulting mixturewas stirred at RT for 18 h. The reaction mixture was added ontosaturated aqueous NaHCO₃ solution (400 ml). The resulting precipitatewas collected by filtration then washed with water and dried undervacuum to afford the intermediate as a light grey solid. The solid thusobtained was added portionwise to acetic acid (150 ml). The resultingsuspension was heated at 100° C. for 2.5 h then allowed to cool to RT.The reaction mixture was concentrated in vacuo then the residue waspartitioned between EtOAc (300 ml) and water (300 ml). The resultingprecipitate was collected by filtration and washed with EtOAc (200 ml)and water (200 ml) then dried under vacuum to afford the product as apink solid (17.9 g, 76%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.96 (dd, J=5.6, 3.0 Hz, 2H), 7.93-7.88 (m,3H), 7.44 (d, J=8.5 Hz, 1H), 7.27 (dd, J=8.5, 1.9 Hz, 1H), 5.12 (s, 2H),4.39 (q, J=7.2 Hz, 2H), 1.37 (t, J=7.2 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=384 [M(⁷⁹Br)H+], 386 [M(⁸¹Br)H+],R_(t)=1.12 min, UV purity=100%.

Intermediate 66—Synthesis of tert-butylN-(3-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}prop-2-yn-1-yl)carbamate

CuI (0.25 g, 1.29 mmol) was added to a solution of2-[(6-bromo-1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione,Intermediate 65 (5.00 g, 13.0 mmol) and tert-butylN-(prop-2-yn-1-yl)carbamate (2.40 g, 15.5 mmol) in DMF (60 ml). Nitrogenwas bubbled through the reaction mixture for 5 min then Pd(PPh₃)₄ (0.74g, 0.64 mmol) was added, followed by triethylamine (2.92 ml, 19.3 mmol).The reaction mixture was heated at 65° C. for 24 h then concentrated invacuo. The crude material was purified by flash column chromatography ona silica column (100 g). The column was eluted with EtOAc:heptane,increasing the gradient linearly from 0:100 to 50:50 over 10 columnvolumes. The desired fractions were combined and evaporated to affordthe product as a yellow foam (2.45 g, 39%).

¹H NMR (250 MHz, DMSO-d₆) δ 7.99-7.88 (m, 4H), 7.71 (s, 1H), 7.46 (d,J=8.3 Hz, 1H), 7.35 (s, 1H), 7.16 (dd, J=8.3, 1.3 Hz, 1H), 5.13 (s, 2H),4.40 (q, J=7.1 Hz, 2H), 4.02-3.97 (m, 2H), 1.41 (s, 9H), 1.36 (t, J=7.1Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=459 [MH⁺], R_(t)=1.17 min, UV purity=95%.

Intermediate 67—Synthesis of tert-butylN-(3-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}propyl)carbamate

Palladium on carbon (10 wt %, 557 mg) was added to a solution oftert-butylN-(3-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}prop-2-yn-1-yl)carbamate,Intermediate 66 (2.4 g, 5.23 mmol) in EtOH (120 ml). The reactionmixture was stirred at RT under a hydrogen atmosphere for 48 h. Thereaction was recharged with palladium on carbon (10 wt %, 278 mg) andstirred at RT under a hydrogen atmosphere for a further 24 h. Thereaction was re-charged with palladium on carbon (10 wt %, 278 mg) andstirred at RT under a hydrogen atmosphere for a further 24 h. Thereaction mixture was filtered through a Celite pad. The Celite pad wasrinsed with EtOH (100 ml), MeOH (100 ml), EtOAc (100 ml), and DMF (5ml). The combined filtrate was concentrated in vacuo then the crudematerial was purified by flash column chromatography on a silica column(25 g). The column was eluted with EtOAc:heptane, increasing thegradient linearly from 0:100 to 75:25 over 10 column volumes. Thedesired fractions were combined and evaporated to afford the product asa light yellow solid (1.20 g, 43%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.98-7.93 (m, 2H), 7.92-7.88 (m, 2H),7.37-7.34 (m, 2H), 6.97 (dd, J=8.2, 1.5 Hz, 1H), 6.84 (t, J=5.3 Hz, 1H),5.09 (s, 2H), 4.35 (q, J=7.2 Hz, 2H), 2.96-2.90 (m, 2H), 2.69-2.63 (m,2H), 1.71 (p, J=7.3 Hz, 2H), 1.40-1.35 (m, 12H).

LC/MS (System A): m/z (ESI⁺)=463 [MH⁺], R_(t)=1.07 min, UV purity=86%.

Intermediate 68—Synthesis of6-(3-{[(tert-butoxy)carbonyl]amino}propyl)-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide

Iodoethane (1.04 ml, 13.0 mmol) was added to a solution of tert-butylN-(3-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}propyl)carbamate,Intermediate 67 (86%, 1.20 g, 2.23 mmol) in MeCN (18 ml) in a pressuretube. The tube was sealed and heated at 110° C. for 4 h. The reactionwas allowed to cool to RT then iodoethane (1.04 ml, 13.0 mmol) was addedthen the reaction was heated at 110° C. for a further 4 h. The reactionmixture was allowed to cool to RT then concentrated in vacuo to affordthe product as a brown solid (1.52 g, >99%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.02 (d, J=8.6 Hz, 1H), 7.97-7.93 (m, 3H),7.92-7.88 (m, 2H), 7.58 (d, J=8.6 Hz, 1H), 6.89 (s, 1H), 5.42 (s, 2H),4.70-4.66 (m, 4H), 2.93 (q, J=6.2 Hz, 2H), 2.79 (t, J=7.4 Hz, 2H),1.81-1.72 (m, 2H), 1.44-1.40 (m, 6H), 1.37 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=491 [M⁺], R_(t)=1.08 min, UV purity=91%.

Intermediate 69—Synthesis of2-(aminomethyl)-6-(3-{[(tert-butoxy)carbonyl]amino}propyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide

Hydrazine hydrate (609 μl, 12.5 mmol) was added to a solution of6-(3-{[(tert-butoxy)carbonyl]amino}propyl)-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 68 (91%, 1.52 g, 2.23 mmol) in MeOH (20 ml) in apressure tube. The tube was sealed then heated at 75° C. for 3 h. Thereaction mixture was allowed to cool to RT then concentrated in vacuo.The residue was suspended in CH₂Cl₂:MeOH (9:1, 20 ml) then filtered. Thefiltrate was concentrated in vacuo to afford the product as a yellowfoam (1.21 g, 89%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.95 (d, J=8.5 Hz, 1H), 7.88 (s, 1H), 7.51(dd, J=8.5, 1.3 Hz, 1H), 6.91 (t, J=5.5 Hz, 1H), 4.60-4.52 (m, 4H), 4.26(s, 2H), 2.93 (app. q, J=6.6 Hz, 2H), 2.78 (t, J=7.5 Hz, 2H), 1.80-1.70(m, 2H), 1.46-1.40 (m, 6H), 1.37 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=361 [M⁺], R_(t)=0.81 min, UV purity=80%.

Intermediate 70—Synthesis of tert-butyl4-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}-1,2,3,6-tetrahydropyridine-1-carboxylate

tert-Butyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate(966 mg, 3.12 mmol) was added to a solution of2-[(6-bromo-1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione,Intermediate 65 (1.00 g, 2.60 mmol) in dioxane (10 ml) and water (2 ml).K₂CO₃ (1.08 g, 7.81 mmol) was added then nitrogen was bubbled throughthe resulting suspension for 10 min. Pd(dppf)₂Cl₂ (190 mg, 0.26 mmol)was added then the reaction mixture was heated under microwaveirradiation for 2 h at 85° C. The reaction mixture was partitionedbetween EtOAc (100 ml) and water (50 ml). The phases were separated thenthe organic phase was washed with brine (50 ml) then dried over Na₂SO₄,filtered and concentrated in vacuo. The crude material was purified byflash column chromatography on a silica column (50 g). The column waseluted with EtOAc:heptane, using the following gradient (% EtOAc, columnvolumes): 0%, 1 CV; 0-50%, 7 CV; 50-79%, 3 CV; 79%, 2 CV; 79-92%, 2 CV,100%, 2 CV. The desired fractions were combined and concentrated invacuo to afford the product as a white solid (670 mg, 50%).

¹H NMR (250 MHz, DMSO-d₆) δ 7.99-7.87 (m, 4H), 7.60 (s, 1H), 7.42 (d,J=8.5 Hz, 1H), 7.25 (dd, J=8.5, 1.5 Hz, 1H), 6.16 (s, 1H), 5.11 (s, 2H),4.49-4.31 (m, 2H), 4.05-3.97 (m, 2H), 3.56 (t, J=5.6 Hz, 2H), 2.54 (s,2H), 1.43 (s, 9H), 1.37 (t, J=7.1 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=487 [MH⁺], R_(t)=1.18 min, UV purity=95%.

Intermediate 71—Synthesis of tert-butyl4-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}piperidine-1-carboxylate

Palladium on carbon (10 wt %, 142 mg) was added to a solution oftert-butyl4-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}-1,2,3,6-tetrahydropyridine-1-carboxylate,Intermediate 70 (95%, 660 mg, 1.29 mmol) in EtOH (40 ml). The reactionmixture was stirred at RT under a hydrogen atmosphere for 16 h. Thereaction was recharged with palladium on carbon (10 wt %, 140 mg) andstirred at RT under a hydrogen atmosphere for a further 48 h. Thereaction mixture was filtered through Celite pad then concentrated invacuo to afford the product as a colourless oil (635 mg, 94%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.96 (dd, J=5.6, 2.9 Hz, 2H), 7.91 (dd,J=5.6, 3.0 Hz, 2H), 7.45 (s, 1H), 7.38 (d, J=8.3 Hz, 1H), 7.02 (dd,J=8.4, 1.4 Hz, 1H), 5.09 (s, 2H), 4.37 (q, J=7.2 Hz, 2H), 4.33 (t, J=5.1Hz, 1H), 4.10 (d, J=10.4 Hz, 2H), 2.84-2.75 (m, 2H), 1.78 (d, J=12.8 Hz,2H), 1.58 (qd, J=12.7, 4.3 Hz, 2H), 1.42 (s, 9H), 1.37 (t, J=7.2 Hz,3H).

LC/MS (System A): m/z (ESI⁺)=489 [MH⁺], R_(t)=1.13 min, UV purity=93%.

Intermediate 72—Synthesis of6-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide

Iodoethane (486 μl, 6.04 mmol) was added to a solution of tert-butyl4-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}piperidine-1-carboxylate,Intermediate 71 (93%, 635 mg, 1.21 mmol) in MeCN (10 ml). The reactionmixture was heated under microwave irradiation for 3 h at 120° C. Thereaction mixture was concentrated in vacuo to afford the product as abrown solid (765 mg, 84%).

¹H NMR (250 MHz, DMSO-d₆) δ 8.07-8.01 (m, 2H), 7.98-7.88 (m, 4H), 7.64(d, J=9.3 Hz, 1H), 5.43 (s, 2H), 4.69 (q, J=7.0 Hz, 4H), 4.20-4.04 (m,2H), 3.05-2.71 (m, 3H), 1.85-1.75 (m, 2H), 1.67 (td, J=12.3, 3.5 Hz,2H), 1.46-1.37 (m, 15H).

LC/MS (System A): m/z (ESI⁺)=517 [M⁺], R_(t)=1.06 min, UV purity=86%.

Intermediate 73—Synthesis of2-(aminomethyl)-6-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide

Hydrazine hydrate (284 μl, 5.82 mmol) was added to a solution of6-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 72 (86%, 750 mg, 1.00 mmol) in MeOH (10 ml) in apressure tube. The tube was sealed then the reaction mixture was heatedat 75° C. for 5 h. The reaction mixture was allowed to cool to RT thenconcentrated in vacuo. The residue was suspended in CH₂Cl₂:MeOH (9:1, 20ml) then filtered. The filtrate was concentrated in vacuo to afford theproduct as a yellow foam (605 mg, 85%).

¹H NMR (250 MHz, DMSO-d₆) δ 7.96 (d, J=8.7 Hz, 1H), 7.95 (s, 1H), 7.58(d, J=8.7 Hz, 1H), 4.65-4.51 (m, 4H), 4.27 (s, 2H), 4.13 (d, J=13.3 Hz,2H), 3.02-2.77 (m, 3H), 1.89-1.76 (m, 2H), 1.72-1.58 (m, 2H), 1.47-1.39(m, 15H).

LC/MS (System A): m/z (ESI⁺)=387 [M⁺], R_(t)=0.89 min, UV purity=72%.

Intermediate 74—Synthesis of tert-butyl4-(4-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}-1H-pyrazol-1-yl)piperidine-1-carboxylate

tert-Butyl4-[4-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]piperidine-1-carboxylate(540 mg, 1.43 mmol) was added to a solution of2-[(6-bromo-1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione,Intermediate 65 (500 mg, 1.30 mmol) in dioxane (10 ml) and water (0.5ml) in a pressure tube. Cs₂CO₃ (848 mg, 2.60 mmol) was added then theresulting suspension was de-gassed by bubbling a stream of nitrogenthrough the reaction mixture for 10 min. XPhos-Pd-G2 (61 mg, 0.078 mmol)was added then the nitrogen bubbling was continued for a further 5 min.The tube was sealed then heated at 100° C. for 16 h. The reactionmixture was concentrated in vacuo then partitioned between EtOAc (100ml) and water (100 ml). The phases were separated then the organic phasewas washed with water (50 ml) and brine (2×50 ml), then dried overMgSO₄, filtered and evaporated. The crude material was purified by flashcolumn chromatography on a silica column (50 g). The column was elutedwith EtOAc:heptane, increasing the gradient linearly from 0:100 to 100:0over 10 column volumes. The desired fractions were combined andevaporated to yield the product as a light yellow foam (226 mg, 31%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.28 (s, 1H), 7.98-7.94 (m, 2H), 7.94-7.89(m, 3H), 7.81-7.76 (m, 1H), 7.43 (d, J=8.4 Hz, 1H), 7.38 (dd, J=8.4, 1.5Hz, 1H), 5.11 (s, 2H), 4.43-4.34 (m, 3H), 4.04 (dd, J=15.8, 8.8 Hz, 2H),2.93 (s, 2H), 2.09-2.02 (m, 2H), 1.81 (qd, J=12.4, 4.3 Hz, 2H), 1.43 (s,9H), 1.42-1.39 (m, 3H).

LC/MS (System A): m/z (ESI⁺)=555 [MH⁺], R_(t)=1.13 min, UV purity=100%.

Intermediate 75—Synthesis of6-(1-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1H-pyrazol-4-yl)-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide

Iodoethane (159 μl, 1.98 mmol) was added to a solution of tert-butyl4-(4-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}-1H-pyrazol-1-yl)piperidine-1-carboxylate,Intermediate 74 (220 mg, 0.397 mmol) in MeCN (5 ml). The reactionmixture was heated under microwave irradiation for 2 h at 120° C.Iodoethane (130 μl, 1.62 mmol) was added then the reaction mixture washeated under microwave irradiation for 1 h at 120° C. The reactionmixture was concentrated in vacuo then azeotroped with diethyl ether.The residue was dried under vacuum to afford the product as a lightyellow solid (280 mg, 80%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.50 (s, 1H), 8.29 (s, 1H), 8.12 (s, 1H),8.10 (d, J=8.8 Hz, 1H), 7.98-7.94 (m, 3H), 7.94-7.89 (m, 2H), 5.42 (s,2H), 4.74-4.65 (m, 4H), 4.48-4.35 (m, 1H), 4.06 (d, 2H), 2.95 (br. s,2H), 2.08 (d, J=6.7 Hz, 2H), 1.81 (qd, J=12.7, 4.7 Hz, 2H), 1.49-1.40(m, 15H).

LC/MS (System A): m/z (ESI⁺)=583 [M⁺], R_(t)=1.13 min, UV purity=81%.

Intermediate 76—Synthesis of2-(aminomethyl)-6-(1-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1H-pyrazol-4-yl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide

Hydrazine hydrate (96 μl, 2.0 mmol) was added to a solution of6-(1-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1H-pyrazol-4-yl)-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 75 (81%, 280 mg, 0.32 mmol) in MeOH (5 ml) in apressure tube. The tube was sealed then heated at 75° C. for 1 h. Thereaction mixture was allowed to cool to RT then concentrated in vacuo.The residue was suspended in CH₂Cl₂ (9:1, 20 ml) then filtered. Thesolid thus obtained was washed further CH₂Cl₂ (20 ml). The filtrateswere combined and evaporated to dryness to afford the product as a lightyellow solid (224 mg, >99%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.48 (s, 1H), 8.23 (s, 1H), 8.10 (s, 1H),8.03 (d, J=8.7 Hz, 1H), 7.90 (dd, J=8.7, 1.3 Hz, 1H), 4.62-4.56 (m, 4H),4.44-4.36 (m, 1H), 4.28 (s, 2H), 4.07 (d, J=10.4 Hz, 2H), 2.94 (br. s,2H), 2.08 (d, J=10.4 Hz, 2H), 1.82 (tt, J=12.2, 6.2 Hz, 2H), 1.46-1.40(m, 15H).

LC/MS (System A): m/z (ESI⁺)=453 [M⁺], R_(t)=0.91 min, UV purity=83%.

Intermediate 77—Synthesis of methyl2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylate

A mixture of 2-{[(tert-butoxy)carbonyl]amino}acetic acid (8.57 g, 48.9mmol), HATU (20.5 g, 53.8 mmol) and DIPEA (17.0 ml, 97.8 mmol) in DMF(200 ml) was stirred at RT for 1 h. Methyl4-amino-3-(ethylamino)benzoate (9.59 g, 48.9 mmol) was added portionwisethen rinsed into the reaction with THF (20 ml). The reaction mixture wasstirred at RT for 18 h. A solution of2-{[(tert-butoxy)carbonyl]amino}acetic acid (0.857 g, 4.89 mmol), HATU(1.86 g, 4.89 mmol) and DIPEA (1.70 ml, 9.78 mmol) in DMF (3 ml) wasstirred at RT for 15 min then added to the main reaction. The resultingsolution was stirred at RT for 3 h. A solution of2-{[(tert-butoxy)carbonyl]amino}acetic acid (0.857 g, 4.89 mmol), HATU(1.86 g, 4.89 mmol) and DIPEA (1.70 ml, 9.78 mmol) in DMF (3 ml) wasstirred at RT for 15 min then added to the main reaction. The resultingsolution was stirred at RT for 64 h. The reaction mixture was added tosaturated aqueous NaHCO₃ solution (200 ml). EtOAc (150 ml) and water(100 ml) were added then the phases were separated. The aqueous phasewas extracted with EtOAc (2×150 ml), then the combined organic phaseswere washed with water (4×100 ml) and brine (50 ml) then dried overNa₂SO₄, filtered and evaporated to afford the crude intermediate as ablack oil (18 g). The oil thus obtained was dissolved in acetic acid (80ml) and stirred at 70° C. for 1 h. The reaction was allowed to cool toRT then evaporated to afford a brown solid. The solid was suspended inEtOAc (200 ml) then filtered and was washed with EtOAc, then dried undervacuum to afford a pale pink solid (6.5 g). The solid thus obtained wassuspended in EtOAc (200 ml). The resulting suspension was heated at 50°C. for 15 min then allowed to cool to RT. The solid was collected byfiltration to afford the product as a white solid (2.43 g). The filtratewas again filtered and the solid was collected by filtration, washedwith EtOAc:heptane then dried under vacuum to afford a second batch ofthe product as a white solid (1.34 g). The filtrate was transferred to aseparating funnel then extracted with saturated aqueous NaHCO₃ solution(3×100 ml), water (100 ml) and brine (50 ml) then dried over Na₂SO₄,filtered and evaporated to a yellow solid which was suspended in theminimum volume of EtOAc:heptane (1:4) and filtered then dried undervacuum to afford a third batch of the product as a white solid (1.77 g).The filtrate from the first filtration was transferred to a separatingfunnel then extracted with saturated aqueous NaHCO₃ solution (3×100 ml),water (100 ml) and brine (50 ml) then dried over Na₂SO₄, filtered andevaporated to a dark brown solid. The solid was suspended in EtOAc (50ml) then filtered. The solid was dried under vacuum to afford a fourthbatch of the product as a white solid (3.4 g). The filtrate wasevaporated to afford a dark solid (8 g). The solid thus obtained wasdissolved in CH₂Cl₂ then evaporated onto silica (16 g). The crudematerial was purified by flash column chromatography on a silica column(100 g). The column was eluted with EtOAc:heptane, increasing thegradient linearly from 0:100 to 100:0 over 10 column volumes. Thedesired fractions were combined and evaporated to afford a brown solid.The solid thus obtained was suspended in EtOAc:heptane (1:4, 20 ml) thenfiltered. The solid was washed with EtOAc:heptane then dried undervacuum to afford a fifth batch of the product as a white solid (1.45 g).The filtrate was concentrated in vacuo then the residue was suspended inEtOAc, filtered and dried under vacuum to afford a sixth batch of theproduct as an off-white solid (0.32 g). The 6 batches of solid werecombined as an EtOAc suspension then evaporated and dried under vacuumto yield the product as an off-white solid (10.7 g, 66%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.16 (d, J=1.1 Hz, 1H), 7.82 (dd, J=8.4, 1.6Hz, 1H), 7.66 (d, J=8.4 Hz, 1H), 7.52 (d, J=5.3 Hz, 1H), 4.47 (d, J=5.8Hz, 2H), 4.35 (q, J=7.2 Hz, 2H), 3.88 (s, 3H), 1.46-1.22 (m, 12H).

LC/MS (System A): m/z (ESI⁺)=334 [MH⁺], R_(t)=0.98 min, UV purity=100%.

Intermediate78—2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylicacid

Aqueous LiOH solution (2.0 M, 16 ml, 32 mmol) was added to a suspensionof methyl2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylate,Intermediate 77 (6.91 g, 20.7 mmol) in THF (100 ml). The reactionmixture was stirred at 50° C. for 16 h then allowed to cool to RT. Thereaction mixture was concentrated in vacuo then the resulting solid wassuspended in water (50 ml). Aqueous HCl solution (2 M) was addeddropwise until pH 4 was reached. The resultant suspension was filteredthen the solid was washed with the minimum of water and MeCN then driedunder vacuum to afford the product as a white solid (6.05 g, 90%).

¹H NMR (500 MHz, DMSO-d₆) δ 12.77 (s, 1H), 8.18-8.07 (m, 1H), 7.80 (dd,J=8.4, 1.5 Hz, 1H), 7.63 (d, J=8.4 Hz, 1H), 7.52 (s, 1H), 4.47 (d, J=5.8Hz, 2H), 4.34 (q, J=7.2 Hz, 2H), 1.46-1.21 (m, 12H).

LC/MS (System A): m/z (ESI⁺)=320 [MH⁺], R_(t)=0.84 min, UV purity=99%.

Intermediate 79—Synthesis of2-(aminomethyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylic aciddihydrochloride

HCl solution in dioxane (4.0 M, 14 ml, 56 mmol) was added to asuspension of2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylicacid, Intermediate 78 (3.55 g, 11.1 mmol) in MeCN (60 ml). The reactionmixture was stirred at RT for 4 h then filtered. The solid was driedunder vacuum to afford the solid as a white solid (3.39 g, 98%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.78 (s, 3H), 8.25 (s, 1H), 7.88 (dd, J=8.5,1.2 Hz, 1H), 7.74 (d, J=8.4 Hz, 1H), 4.50 (d, J=4.4 Hz, 2H), 4.38 (q,J=7.2 Hz, 2H), 1.34 (t, J=7.2 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=220 [MH⁺], R_(t)=0.16 min, ELS purity=94%.

Intermediate 80—Synthesis of2-({[(benzyloxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylicacid

NaHCO₃ (4.83 g, 57.5 mmol) was added portionwise to a cooled (0° C.)suspension of 2-(aminomethyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylicacid dihydrochloride, Intermediate 79 (4.20 g, 14.4 mmol) in water (40ml). The reaction mixture was allowed to warm to RT then a solution ofbenzyl 2,5-dioxopyrrolidin-1-yl carbonate (3.94 g, 15.8 mmol) in THF (40ml) was added dropwise over 15 min. The reaction mixture was left tostir at RT for 16. The resultant mixture was extracted with EtOAc (50ml). The phases were separated then the organic phase was washed withwater (3×10 ml). The combined aqueous phases were acidified to pH 5 byaddition of aqueous HCl solution (2 M), resulting in precipitation of asolid. The resultant suspension was filtered then the solid was driedunder vacuum to afford the product as a white solid (3.5 g, 69%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.12 (s, 1H), 7.97 (t, J=5.8 Hz, 1H), 7.80(dd, J=8.4, 1.4 Hz, 1H), 7.62 (d, J=8.4 Hz, 1H), 7.42-7.09 (m, 5H), 5.06(s, 2H), 4.54 (d, J=5.9 Hz, 2H), 4.33 (q, J=7.0 Hz, 2H), 1.28 (t, J=7.1Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=354 [MH⁺], R_(t)=0.89 min, UV purity=100%.

Intermediate 81—tert-butyl2-({[(benzyloxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylate

1,1-Di-tert-butoxy-N,N-dimethylmethanamine (6.77 ml, 28.3 mmol) wasadded to a suspension of2-({[(benzyloxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylicacid, Intermediate 80 (2.50 g, 7.08 mmol) in α,α,α-trifluorotoluene (50ml). The reaction mixture was heated at 100° C. for 1 h. The reactionmixture was allowed to cool to RT then1,1-di-tert-butoxy-N,N-dimethylmethanamine (6.77 ml, 28.3 mmol) wasadded dropwise over 15 min. The resultant mixture was heated at 100° C.for 45 min. The reaction mixture was cooled to 50° C. then1,1-di-tert-butoxy-N,N-dimethylmethanamine (3.38 ml, 14.15 mmol) wasadded dropwise over 5 min. The resultant mixture was heated at 100° C.for 0.5 h then allowed to cool to RT. The reaction mixture waspartitioned between EtOAc (50 ml) and water (50 ml). The phases wereseparated then the organic phase was washed with water (2×30 ml),saturated aqueous NaHCO₃ solution (20 ml) and brine (10 ml) then driedover Na₂SO₄, filtered and concentrated in vacuo to afford a beige solid(2.5 g). The solid thus obtained was suspended in MeCN (10 ml). Thesolid was collected by filtration then dried under vacuum to afford theproduct as an off-white solid (2.30 g, 79%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.07 (s, 1H), 7.97 (m, 1H), 7.76 (dd, J=8.4,1.5 Hz, 1H), 7.64 (d, J=8.4 Hz, 1H), 7.34 (m, 5H), 5.07 (s, 2H), 4.55(d, J=6.0 Hz, 2H), 4.38-4.25 (m, 2H), 1.57 (s, 9H), 1.29 (m, 3H).

LC/MS (System A): m/z (ESI⁺)=410 [MH⁺], R_(t)=1.17 min, UV purity=99%.

Intermediate 82—Synthesis of2-({[(benzyloxy)carbonyl]amino}methyl)-6-[(tert-butoxy)carbonyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide

Three reactions were run independently as follows then combined forwork-up. Reaction 1: a suspension of tert-butyl2-({[(benzyloxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylate,Intermediate 81 (800 mg, 1.95 mmol) and iodoethane (629 μl, 7.82 mmol)in MeCN (10 ml) was heated under microwave irradiation for 2 h at 120°C. The reaction was retreated with iodoethane (629 μl, 7.82 mmol) thenthe reaction mixture was heated under microwave irradiation for afurther 2 h at 120° C. Reaction 2: a suspension of tert-butyl2-({[(benzyloxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylate,Intermediate 81 (800 mg, 1.95 mmol) and iodoethane (629 μl, 7.82 mmol)in MeCN (10 ml) was heated under microwave irradiation for 1 h 45 min at120° C. The reaction was retreated with iodoethane (629 μl, 7.82 mmol)then the reaction mixture was heated under microwave irradiation for afurther 1.5 h at 120° C. Reaction 3: a suspension of tert-butyl2-({[(benzyloxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylate,Intermediate 81 (700 mg, 1.71 mmol) and iodoethane (591 μl, 6.84 mmol)in MeCN (10 ml) was heated under microwave irradiation for 1.5 h at 120°C. The reaction was retreated with iodoethane (629 μl, 7.82 mmol) thenthe reaction mixture was heated under microwave irradiation for afurther 1.5 h at 120° C. The three reactions were combined andconcentrated in vacuo. The crude material was purified by flash columnchromatography on C18 (60 g). The column was eluted with MeCN:water+0.1%formic acid using the following gradient (% MeCN, column volumes): 5%, 2CV; 5-31%, 5 CV; 31%, 4 CV; 31-59%, 6 CV; 59-100%, 3 CV; 100% 1 CV. Thedesired fractions were combined and concentrated in vacuo to afford theproduct as a white foam (2.13 g, 67%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.54 (s, 1H), 8.43 (t, J=5.1 Hz, 1H),8.22-8.12 (m, 2H), 7.42-7.27 (m, 5H), 5.06 (s, 2H), 4.90 (d, J=5.3 Hz,2H), 4.79-4.59 (m, 4H), 1.61 (s, 9H), 1.47-1.36 (m, 6H).

LC/MS (System A): m/z (ESI⁺)=438 [M⁺], R_(t)=1.07 min, UV purity=100%.

Intermediate 83—Synthesis of2-(aminomethyl)-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrobromide bromide

HBr solution in AcOH (33 wt %, 4.28 ml, 18.8 mmol) was added to asolution of2-({[(benzyloxy)carbonyl]amino}methyl)-6-[(tert-butoxy)carbonyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 82 (2.13 g, 3.77 mmol) in AcOH (10 ml). Thereaction mixture was stirred at RT for 0.5 h. The resultant suspensionwas concentrated in vacuo then azeotroped with MeCN. The solid thusobtained was suspended in the minimum volume of MeCN then filtered anddried under vacuum to afford the product as a white solid (1.52 g, 99%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.69 (s, 1H), 8.31-8.20 (m, 2H), 4.85-4.63(m, 6H), 1.53-1.40 (m, 6H).

LC/MS (System A): m/z (ESI⁺)=248 [M⁺], R_(t)=0.15 min, ELS purity=100%.

Intermediate 84—Synthesis of3-amino-N-[(1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-5H-pyrrolo[2,3-b]pyrazine-2-carboxamide

(1-Ethyl-1H-1,3-benzodiazol-2-yl)methanaminium chloride (380 mg, 1.79mmol) was added to a solution of lithium(1⁺) ion3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate 3 (300 mg,1.63 mmol), HBTU (927 mg, 2.44 mmol) and DIPEA (852 μl, 4.89 mmol) inDMF (4 ml). The reaction mixture was stirred at RT for 20 h. AdditionalHBTU (450 mg, 1.18 mmol) was added and the reaction was left to stir atRT for a further 5 h. The reaction mixture was partitioned between water(100 ml) and EtOAc (80 ml). The phases were separated then the aqueousphase was extracted with EtOAc (2×80 ml). The combined organic phaseswere dried over Na₂SO₄ then concentrated in vacuo. The crude materialwas purified by flash column chromatography on C18 (60 g). The columnwas eluted with MeCN:water+0.1% NH₄OH using the following gradient (%MeCN, column volumes): 10%, 2 CV; 10-45%, 15 CV; 45-90%, 4 CV; 90%, 2CV. The desired fractions were combined and concentrated in vacuo toafford the product as a yellow solid (78 mg, 11%).

1H NMR (500 MHz, DMSO-d₆) δ 11.46 (s, 1H), 9.08 (t, J=5.6 Hz, 1H), 7.60(d, J=7.7 Hz, 1H), 7.55 (d, J=7.9 Hz, 1H), 7.48 (d, J=3.7 Hz, 1H),7.38-7.10 (m, 4H), 6.44 (d, J=3.7 Hz, 1H), 4.80 (d, J=5.6 Hz, 2H), 4.33(q, J=7.2 Hz, 2H), 1.32 (t, J=7.2 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=336 [MH⁺], R_(t)=0.87 min, UV purity=79%.

Intermediate 85—Synthesis of3-amino-N-[(1-benzyl-1H-1,3-benzodiazol-2-yl)methyl]-5H-pyrrolo[2,3-b]pyrazine-2-carboxamide

A mixture of lithium(1⁺) ion3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate 3 (50 mg,0.27 mmol), HBTU (113 mg, 0.272 mmol) and DIPEA (142 μl, 0.815 mmol) inDMF (1 ml) was stirred at RT for 1 h.(1-Benzyl-1H-1,3-benzodiazol-2-yl)methanaminium chloride (82 mg, 0.30mmol) was added then the resulting mixture was stirred at RT for 4 h.Additional HBTU (60 mg, 0.16 mmol) was added and the reaction wasstirred at RT for a further 16 h. The reaction mixture was partitionedbetween water (10 ml) and EtOAc (10 ml). The phases were separated thenthe aqueous phase was extracted with EtOAc (2×10 ml). The combinedorganic phases were dried over Na₂SO₄ then concentrated in vacuo. Thecrude material was purified by flash column chromatography on C18 (30g). The column was eluted with MeCN:water+0.1% NH₄OH using the followinggradient (% MeCN, column volumes): 10%, 2 CV; 10-33%, 11 CV; 33-58%, 6CV. The desired fractions were combined and concentrated in vacuo toafford the product as a yellow solid (39 mg, 35%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.43 (s, 1H), 9.06 (t, J=5.6 Hz, 1H),7.66-7.59 (m, 1H), 7.51-7.45 (m, 2H), 7.31-7.12 (m, 9H), 6.43 (d, J=3.7Hz, 1H), 5.60 (s, 2H), 4.77 (d, J=5.6 Hz, 2H).

LC/MS (System A): m/z (ESI⁺)=398 [MH⁺], R_(t)=0.97 min, UV purity=96%.

Intermediate 86—Synthesis of5-(2-aminoethoxy)-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-iumhydrochloride chloride

HCl solution in dioxane (4.0 M, 14 ml, 56 mmol) was added to a mixtureof6-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)-1,3-diethyl-2-({[(9H-fluoren-9-ylmethoxy)carbonyl]amino}methyl)-1H-1,3-benzodiazol-3-iumiodide, Intermediate 58 (4.28 g, 6.01 mmol) in MeCN (50 ml). Theresulting mixture was stirred at RT for 20 min then concentrated invacuo to afford the product as a brown/orange foam (3.87 g, 98%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.30 (t, J=5.3 Hz, 1H), 8.15 (s (br), 3H),7.99 (d, J=9.1 Hz, 1H), 7.86 (d, J=7.5 Hz, 2H), 7.69-7.60 (m, 3H),7.38-7.30 (m, 3H), 7.25 (t, J=7.3 Hz, 2H), 4.75 (d, J=5.2 Hz, 2H),4.58-4.45 (m, 6H), 4.34 (t, J=4.9 Hz, 2H), 4.22 (t, J=6.0 Hz, 1H),1.37-1.30 (m, 6H).

LC/MS (System A): m/z (ESI⁺)=485 [M⁺], R_(t)=0.84 min, UV purity=85%.

Intermediate 87—Synthesis of5-(2-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}ethoxy)-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-iumchloride

A mixture of 4,6-O-benzylidene-D-glucopyranose (95%, 6.67 g, 23.6 mmol),5-(2-aminoethoxy)-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-iumhydrochloride chloride, Intermediate 86 (85%, 3.87 g, 5.91 mmol) andAcOH (1.35 ml, 23.6 mmol) in MeOH (100 ml) was stirred at RT for 0.5 h.NaCNBH₃ (1.48 g, 23.6 mmol) was added then the resulting mixture wasstirred at RT for 20 h. More MeOH (40 ml) was added then the reactionwas left to stir at RT for a further 24 h. More MeOH (80 ml) was added,then the reaction was retreated with 4,6-O-benzylidene-D-glucopyranose(95%, 1.60 g, 5.67 mmol), AcOH (0.34 ml, 5.94 mmol) and NaCNBH₃ (0.38 g,6.05 mmol). The reaction was left to stir at RT for a further 92 h thenadded to saturated aqueous NaHCO₃ solution (250 ml). The resultantsuspension was stirred at RT for 20 min. The solid was collected byfiltration then washed with water and dried under vacuum to afford theproduct as a pale pink solid (6.43 g, 89%).

LC/MS (System A): m/z (ESI⁺)=990 [M⁺], 496 [(M⁺)⁺H+], R_(t)=0.93 min, UVpurity=84%.

Intermediate88—2-(aminomethyl)-5-(2-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}ethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumchloride

Morpholine (4.77 ml, 55.1 mmol) was added to a stirred mixture of5-(2-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}ethoxy)-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-iumchloride, Intermediate 87 (84% 6.42 g, 5.26 mmol) in THF (60 ml). Theresulting mixture was stirred at RT for 4 h. The reaction mixture wasdiluted with diethyl ether (150 ml). The resulting suspension wasagitated then the suspension was decanted off, leaving behind a viscousoil. More diethyl ether (80 ml) was added to the oil residue then themixture was sonicated. The resulting suspension was again decanted offto leave behind a viscous oil. The process was repeated once more withdiethyl ether (80 ml) then the resulting viscous oil was dried undervacuum to afford the product as a pale purple foam (4.39 g, 85%).

LC/MS (System A): m/z (ESI⁺)=767 [M⁺], 384 [(M⁺)⁺H+], R_(t)=0.75 min, UVpurity=82%.

Intermediate 89—Synthesis of2-(aminomethyl)-5-(2-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}ethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumdihydrochloride chloride

A mixture of2-(aminomethyl)-5-(2-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}ethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumchloride, Intermediate 88 (82%, 1.50 g, 1.53 mmol) and aqueous HClsolution (2.0 M, 25 ml, 50 mmol) was stirred at RT for 1 h. The reactionmixture was concentrated in vacuo then the residue was dissolved inwater (10 ml) and lyophilised to afford the product as a pale purplefoam (1.53 g, >99%).

LC/MS (System A): m/z (ESI⁺)=591 [M⁺], 296 [(M⁺)⁺H+], R_(t)=0.13 min, UVpurity=70%.

Intermediate 90—Synthesis of5-(3-aminopropoxy)-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-iumhydrochloride chloride

HCl solution in dioxane (4.0 M, 3.3 ml, 13 mmol) was added to a mixtureof6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1,3-diethyl-2-({[(9H-fluoren-9-ylmethoxy)carbonyl]amino}methyl)-1H-1,3-benzodiazol-3-iumiodide, Intermediate 63 (95%, 1.00 g, 1.31 mmol) in MeCN (15 ml). Thereaction mixture was stirred at RT for 0.5 h then concentrated in vacuoto afford the product as a viscous yellow oil (875 mg, >99% —yieldcorrected for 15 wt % residual dioxane observed in NMR).

¹H NMR (500 MHz, DMSO-d₆) δ 8.30 (t, J=5.3 Hz, 1H), 8.00-7.85 (m, 6H),7.65-7.59 (m, 3H), 7.37 (t, J=7.4 Hz, 2H), 7.31 (dd, J=9.1, 2.1 Hz, 1H),7.27 (t, J=7.4 Hz, 2H), 4.76 (d, J=5.4 Hz, 2H), 4.58-4.46 (m, 6H),4.27-4.19 (m, 3H), 3.05-2.95 (m, 2H), 2.12-2.06 (m, 2H), 1.34 (t, J=7.1Hz, 6H).

LC/MS (System A): m/z (ESI⁺)=499 [M⁺], R_(t)=0.89 min, UV purity=98%.

Intermediate 91—Synthesis of5-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propoxy)-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-iumhydrochloride chloride

A mixture of 4,6-O-benzylidene-D-glucopyranose (1.43 g, 5.32 mmol),5-(3-aminopropoxy)-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-iumhydrochloride chloride, Intermediate 90 (85%, 875 mg, 1.30 mmol) andAcOH (305 μl, 5.32 mmol) in MeOH (25 ml) was stirred at RT for 0.5 h.NaCNBH₃ (334 mg, 5.32 mmol) was added then the resulting mixture wasstirred at RT for 64 h. Additional 4,6-O-benzylidene-D-glucopyranose(500 mg, 1.86 mmol) and AcOH (110 μl, 1.92 mmol) was added. The mixturewas stirred for 0.5 h then NaCNBH₃ (115 mg, 1.83 mmol) was added. Theresulting mixture was stirred at RT for a further 16 h. Saturatedaqueous NaHCO₃ solution (40 ml) was added over 5 min whereupon a whiteprecipitate formed. The resultant suspension was filtered and thecollected solid was washed with water then dried under vacuum to affordthe product as a white solid (1.39 g, 60%).

LC/MS (System A): m/z (ESI⁺)=503 [(M⁺)⁺H+], R_(t)=1.00 min, UVpurity=60%.

Intermediate 92—Synthesis of2-(aminomethyl)-5-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrochloride chloride

Morpholine (659 μl, 7.62 mmol) was added to a mixture of5-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propoxy)-1,3-diethyl-2-({[(9H-fluoren-9-ylmethoxy)carbonyl]amino}methyl)-1H-1,3-benzodiazol-3-iumchloride hydrochloride, Intermediate 91 (60%, 1.32 g, 0.762 mmol) in THF(10 ml). The mixture was stirred at RT for 2.5 h. The reaction mixturewas diluted with diethyl ether (20 ml). The resulting suspension wassonicated then the suspension was decanted off, leaving behind a viscousoil. More diethyl ether (20 ml) was added to the oil residue then themixture was sonicated. The resulting suspension was again decanted offto leave behind a viscous oil. The process was repeated once more withdiethyl ether (20 ml) then the resulting viscous oil was dried undervacuum to afford the product as a pale orange solid (639 mg, 79%).

LC/MS (System A): m/z (ESI⁺)=781 [M⁺], 391 [(M⁺)⁺H+], R_(t)=0.78 min, UVpurity=80%.

Intermediate 93—Synthesis of2-(aminomethyl)-5-(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrochloride chloride

A mixture of2-(aminomethyl)-5-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrochloride chloride, Intermediate 92 (80%, 375 mg, 0.367 mmol) andaqueous HCl solution (2.0 M, 8.0 ml, 16 mmol) was stirred at RT for 40min. The reaction mixture was concentrated in vacuo then diluted withwater and lyophilised to afford the product as a pale orange solid (305mg, 98%).

LC/MS (System A): m/z (ESI⁺)=605 [M⁺], 303 [(M⁺)⁺H+], R_(t)=0.13 min, UVpurity=80%.

Intermediate 94—Synthesis of tert-butyl4-{1-ethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-6-yl}piperidine-1-carboxylate

Step 1: A suspension of hydrazine hydrate (1.27 ml, 1.31 mmol) andtert-butyl4-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}piperidine-1-carboxylateIntermediate 71 (2.56 g, 5.24 mmol) in MeOH (30 ml) was heated underreflux for 2.5 h then allowed to cool to RT. The resultant suspensionwas left to stand at RT for 16 h then filtered. The collected solid waswashed solid with CH₂Cl₂ then the filtrate was concentrated in vacuo andazeotroped with MeCN to afford the intermediate as a white solid (1.24g). Step 2: A solution of (2,5-dioxopyrrolidin-1-yl)9H-fluoren-9-ylmethyl carbonate (1.16 g, 3.43 mmol) in MeCN (20 ml) wasadded dropwise over 10 min to a mixture of the intermediate from Step 1and NaHCO₃ (576 mg, 6.86 mmol) in MeCN (40 ml) and water (30 ml). Thereaction mixture was stirred at RT for 21 h then partitioned betweenEtOAc (100 ml) and water (100 ml). The phases were separated then theorganic phase was washed with water (2×50 ml), brine (50 ml), then driedover Na₂SO₄, filtered and concentrated in vacuo. The solid thus obtainedwas suspended in MeCN (150 ml) under reflux, with sonication at variousintervals. The resultant suspension was allowed to cool to RT thenfiltered. The collected solid was dried under vacuum to afford theproduct as a white solid (1.52 g, 50% over 2 steps).

¹H NMR (500 MHz, DMSO-d₆) δ 7.99 (s, 1H), 7.88 (d, J=7.5 Hz, 2H), 7.72(d, J=7.4 Hz, 2H), 7.48 (d, J=8.2 Hz, 1H), 7.44-7.37 (m, 3H), 7.31 (t,J=7.4 Hz, 2H), 7.07 (d, J=8.3 Hz, 1H), 4.48 (d, J=5.7 Hz, 2H), 4.32 (d,J=7.1 Hz, 2H), 4.23 (t, J=6.9 Hz, 3H), 4.15-4.05 (m, 2H), 2.95-2.75 (m,3H), 1.80 (d, J=12.0 Hz, 2H), 1.65-1.52 (m, 2H), 1.42 (s, 9H), 1.26 (t,J=7.1 Hz, 3H).

LC/MS (System A): m/z (ESI⁺)=581 [MH⁺], R_(t)=1.12 min, UV purity=100%.

Intermediate 95—Synthesis of6-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-iumiodide

A suspension of tert-butyl4-{1-ethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-6-yl}piperidine-1-carboxylate,Intermediate 94 (1.55 g, 2.67 mmol) and iodoethane (1.07 ml, 13.4 mmol)in MeCN (20 ml) was heated under microwave irradiation for 2.5 h at 120°C. The reaction mixture was concentrated in vacuo then the crudematerial was purified by flash column chromatography on a silica column(25 g). The column was eluted with CH₂Cl₂:MeOH, using the followinggradient (% MeOH, column volumes): 0%, 1 CV; 0-2.3%, 5 CV; 2.3%, 2 CV;2.3-10%, 7 CV, 10%, 7 CV. The desired fractions were combined andevaporated to afford the product as a light pink foam (1.60 g, 75%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.26 (t, J=5.2 Hz, 1H), 7.98 (d, J=9.1 Hz,2H), 7.86 (d, J=7.5 Hz, 2H), 7.62 (d, J=7.7 Hz, 3H), 7.35 (t, J=7.4 Hz,2H), 7.25 (t, J=7.3 Hz, 2H), 4.76 (d, J=5.2 Hz, 2H), 4.59-4.44 (m, 6H),4.22 (t, J=5.6 Hz, 1H), 4.16-4.10 (m, 2H), 3.00-2.80 (m, 3H), 1.83 (d,J=12.1 Hz, 2H), 1.66 (qd, J=12.5, 4.0 Hz, 2H), 1.44 (s, 9H), 1.34 (t,J=7.0 Hz, 6H).

LC/MS (System A): m/z (ESI⁺)=609 [M⁺], R_(t)=1.11 min, UV purity=92%.

Intermediate 96—Synthesis of1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-6-(piperidin-4-yl)-1H-1,3-benzodiazol-3-iumhydrochloride iodide

HCl solution in dioxane (4.0 M, 2.0 ml, 8.0 mmol) was added to asolution of6-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-iumiodide, Intermediate 95 (1.59 g, 1.99 mmol) in MeCN (50 ml). Thereaction mixture was stirred at RT for 2 h then concentrated in vacuoand azeotroped with MeCN (3×25 ml) to afford the product as a yellowsolid (1.34 g, >99%).

¹H NMR (500 MHz, DMSO-d₆) δ 9.00 (br.s, 1H), 8.87 (br.s, 1H), 8.35 (t,J=5.1 Hz, 1H), 8.04 (d, J=8.6 Hz, 1H), 7.88 (s, 1H), 7.85 (d, J=7.6 Hz,2H), 7.62 (d, J=7.5 Hz, 2H), 7.58 (d, J=8.5 Hz, 1H), 7.34 (t, J=7.4 Hz,2H), 7.24 (t, J=7.4 Hz, 2H), 4.77 (d, J=5.1 Hz, 2H), 4.61-4.50 (m, 4H),4.48 (d, J=5.9 Hz, 2H), 4.21 (t, J=5.8 Hz, 1H), 3.42 (d, 1H), 3.15-2.96(m, 4H), 2.04-1.91 (m, 4H), 1.34 (t, J=7.1 Hz, 6H).

LC/MS (System A): m/z (ESI⁺)=509 [M⁺], R_(t)=0.85 min, UV purity=100%.

Intermediate 97—Synthesis of6-{1-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]piperidin-4-yl}-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-iumiodide

A solution of1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-6-(piperidin-4-yl)-1H-1,3-benzodiazol-3-iumhydrochloride iodide, Intermediate 96 (1.33 g, 1.98 mmol),4,6-O-benzylidene-D-glucopyranose (1.06 g, 3.95 mmol) and AcOH (226 μl,3.95 mmol) in MeOH (50 ml) was stirred at RT for 0.5 h. NaCNBH₃ (248 mg,3.95 mmol) was added then the resultant solution was stirred at RT for24 h. The reaction mixture was recharged with4,6-O-benzylidene-D-glucopyranose (1.06 g, 3.95 mmol) and AcOH (226 μl,3.95 mmol) then left to stir at RT for 0.5 h. NaCNBH₃ (150 mg, 2.37mmol) was added then the resultant solution was stirred at RT for afurther 16 h. Saturated aqueous NaHCO₃ solution (50 ml) was added over 5min then the resultant suspension was allowed to stand at RT for 0.5 hthen filtered. The collected solid was washed with water (100 ml) thendried under vacuum for afford the product as a beige solid (1.24 g,57%).

LC/MS (System A): m/z (ESI⁺)=381 [(M⁺)⁺H+], R_(t)=0.94 min, UVpurity=81%.

Intermediate 98—Synthesis of2-(aminomethyl)-6-{1-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]piperidin-4-yl}-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide

Morpholine (370 μl, 11.3 mmol) was added to a suspension of6-{1-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]piperidin-4-yl}-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-iumiodide, Intermediate 97 (81% 1.00 g, 0.90 mmol) in THF (10 ml). Theresulting suspension was sonicated for 10 min then left to stir at RTfor 2.5 h. The reaction mixture was diluted with diethyl ether (25 ml).The resulting white precipitate was filtered and washed with diethylether (25 ml) then dried under vacuum to afford the product as a whitesolid (615 mg, 92%).

LC/MS (System A): m/z (ESI⁺)=539 [M⁺], R_(t)=0.73 min, UV purity=90%.

Intermediate 99—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{1-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]piperidin-4-yl}-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide

Imidazole hydrochloride (55 mg, 0.53 mmol) was added to a solution of2-(aminomethyl)-6-{1-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]piperidin-4-yl}-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 98 (352 mg, 0.475 mmol) and2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 4 (181 mg, 0.790 mmol) in DMF (10 ml). The reaction mixturewas stirred at RT for 20 h then concentrated in vacuo. The crudematerial thus obtained was purified by flash column chromatography onC18 (30 g). The column was eluted with MeCN:water+0.1% TFA using thefollowing gradient (% MeCN, column volumes): 0%, 2 CV; 0-6%, 4 CV;6-20%, 7 CV; 20-24%, 1 CV; 24-33%, 1 CV; 33%, 1 CV; 33-43%, 1 CV;43-100%, 2 CV. The desired fractions were combined and concentrated invacuo to afford the product as a yellow solid (401 mg, 99%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.54 (s, 1H), 9.72 (t, J=5.3 Hz, 1H), 9.16(s, 1H), 8.04 (d, J=8.7 Hz, 1H), 7.87 (s, 1H), 7.56 (d, J=8.8 Hz, 1H),7.52 (dd, J=3.7, 2.6 Hz, 1H), 7.49-7.45 (m, 2H), 7.42-7.35 (m, 3H), 6.42(dd, J=3.8, 1.7 Hz, 1H), 5.57 (s, 1H), 5.06 (d, J=5.2 Hz, 2H), 4.77-4.63(m, 4H), 4.23-4.10 (m, 3H), 3.87 (d, J=5.9 Hz, 1H), 3.85-3.64 (m, 4H),3.59 (dt, J=20.8, 10.7 Hz, 4H), 3.38 (d, J=12.5 Hz, 1H), 3.25 (d, J=10.2Hz, 2H), 3.07 (t, J=11.4 Hz, 2H), 2.19-1.85 (m, 4H), 1.41 (td, J=7.2,2.4 Hz, 6H).

LC/MS (System A): m/z (ESI⁺)=350 [(M*)⁺H*], R_(t)=0.81 min, UVpurity=97%.

Intermediate 100—Synthesis of tert-butylN-[3-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propyl]carbamate

A solution of 2,5-dioxopyrrolidin-1-yl 9H-fluoren-9-ylmethyl carbonate(3.87 g, 11.5 mmol) in MeCN (30 ml) was added dropwise over 20 min to amixture of NaHCO₃ (1.93 g, 23.0 mmol) and tert-butylN-(3-aminopropyl)carbamate (2.00 g, 11.5 mmol) in MeCN (40 ml) and water(40 ml). The resultant mixture was stirred at RT for 1 h then filtered.The collected solid was washed with water (2×20 ml) then MeCN (2×20 ml),then dried under vacuum to afford a white solid (1.28 g). The solid thusobtained was suspended in EtOAc (10 ml) then filtered. The solidcollected was dried under vacuum to afford a first batch of the productas a white solid (1.24 g). The MeCN/water filtrate was concentrated invacuo then the resulting residue was partitioned between EtOAc (100 ml)and water (50 ml). The phases were separated then the organic phase waswashed with water (2×50 ml), brine (20 ml), then dried over Na₂SO₄,filtered and concentrated in vacuo to afford a white solid (2.30 g). Thesolid thus obtained was combined with the filtrate from the EtOActrituration then the combined material was purified by flash columnchromatography on a silica column (25 g). The column was eluted withheptane:EtOAc:MeOH using the following gradient: 100:0:0, 3 CV;100:0:0-81:19:0, 3 CV; 81:19:0%, 2 CV; 81:19:0-61:39:0, 3 CV; 61:39:0, 5CV; 61:39:0-12:88:0, 8 CV; 12:88:0-0:100:0, 2 CV; 0:100:0, 1 CV;0:100:0-0:93:7, 4 CV; 0:93:7, 3 CV; 0:93:7-0:91:9, 1 CV. The desiredfractions were combined and evaporated to afford a second batch of theproduct as a white solid (2.60 g), which was analytically identical tothe first batch. Overall yield=3.84 g (84%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.89 (d, J=7.5 Hz, 2H), 7.73-7.59 (m, 2H),7.41 (t, J=7.4 Hz, 2H), 7.36-7.29 (m, 2H), 7.22 (t, J=5.6 Hz, 1H), 6.74(s, 1H), 4.29 (d, J=6.9 Hz, 2H), 4.21 (t, J=6.8 Hz, 1H), 3.04-2.78 (m,4H), 1.56-1.43 (m, 2H), 1.37 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=419 [(M⁺Na)⁺], R_(t)=1.25 min, UVpurity=99%.

Intermediate 101—Synthesis of (9H-fluoren-9-yl)methylN-(3-aminopropyl)carbamate hydrochloride

HCl solution in dioxane (4.0 M, 8.0 ml, 32 mmol) was added to asuspension of tert-butylN-(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propyl)carbamate,Intermediate 100 (2.60 g, 6.56 mmol) in MeCN (40 ml). The reactionmixture was stirred at RT for 1 h then filtered. The collected solid wasrinsed with MeCN then dried under vacuum to afford the product as awhite solid (1.89 g, 87%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.95-7.77 (m, 5H), 7.68 (d, J=7.4 Hz, 2H),7.48-7.38 (m, 3H), 7.37-7.29 (m, 2H), 4.33 (d, J=6.8 Hz, 2H), 4.26-4.17(m, 1H), 3.10-2.99 (m, 2H), 2.81-2.71 (m, 2H), 1.76-1.64 (m, 2H).

LC/MS (System A): m/z (ESI⁺)=297 [MH⁺], R_(t)=0.91 min, UV purity=100%.

Intermediate 102—Synthesis of (9H-fluoren-9-yl)methylN-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propyl)carbamate;formic acid

A mixture of 9H-fluoren-9-ylmethyl N-(3-aminopropyl)carbamatehydrochloride, Intermediate 101 (900 mg, 2.70 mmol) and4,6-O-benzylidene-D-glucopyranose (1.45 g, 5.41 mmol) in MeOH (40 ml)was stirred at RT for 1.5 h. AcOH (0.31 ml, 5.4 mmol) and NaCNBH₃ (340mg, 5.41 mmol) were added then the reaction was stirred at RT for 18 h.The reaction was recharged with 4,6-O-benzylidene-D-glucopyranose (1.45g, 5.41 mmol) then the reaction was stirred at RT for 1 h. NaCNBH₃ (340mg, 5.41 mmol) was added then the reaction was left to stir at RT for afurther 114 h. Saturated aqueous NaHCO₃ solution (50 ml) was addeddropwise over 10 min then the resultant mixture was partitioned betweenEtOAc (50 ml) and water (50 ml). The phases were separated then theorganic phase was washed with saturated aqueous NaHCO₃ solution (2×50ml), water (50 ml) and brine (20 ml), then dried using Na₂SO₄, filteredand concentrated in vacuo to afford a white solid (2.25 g). The crudematerial thus obtained was purified by flash column chromatography onC18 (120 g). The column was eluted with MeCN:water+0.1% formic acidusing the following gradient (% MeCN, column volumes): 10%, 2 CV;10-29%, 4 CV; 29-39%, 2 CV; 39%, 2 CV; 39-47%, 1 CV; 47-73%, 1 CV;73-100%, 1 CV; 100% 1 CV. The desired fractions were combined andconcentrated in vacuo to afford the product as a white solid (1.12 g,49%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.16 (m, 1H), 7.88 (d, J=7.5 Hz, 2H), 7.67(d, J=7.4 Hz, 2H), 7.45-7.37 (m, 6H), 7.36-7.25 (m, 8H), 7.24-7.16 (m,1H), 5.49-5.39 (m, 2H), 5.27-5.01 (m, 2H), 4.32-4.25 (m, 2H), 4.23-4.16(m, 1H), 4.16-4.09 (m, 2H), 3.84-3.74 (m, 4H), 3.73-3.66 (m, 2H),3.64-3.56 (partially obscured m, 2H), 3.53-3.45 (obscured m, 2H),2.99-2.89 (obscured m, 2H), 2.68-2.54 (obscured m, 6H), 1.60-1.48 (m,2H).

LC/MS (System A): m/z (ESI⁺)=801 [MH⁺], R_(t)=1.01 min, UV purity=100%.

Intermediate 103—Synthesis of(1R,2S)-3-[(3-aminopropyl)[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino]-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol;bis(formic acid)

Diethylamine (1.44 ml, 14.0 mmol) was added to a solution of9H-fluoren-9-ylmethylN-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propyl)carbamate;formic acid, Intermediate 102 (1.32 g, 1.40 mmol) in THF (20 ml). Thereaction mixture was stirred at RT for 16 h then concentrated in vacuo.The crude material thus obtained was purified by flash columnchromatography on C18 (60 g). The column was eluted with MeCN:water+0.1%formic acid using the following gradient (% MeCN, column volumes): 5%, 2CV; 5-18%, 3 CV; 18%, 2 CV; 18-26%, 2 CV; 26-100%, 2 CV; 100% 1 CV. Thedesired fractions were combined and concentrated in vacuo to afford theproduct as a white solid (710 mg, 80%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.36-8.24 (m, 2H), 7.44-7.38 (m, 4H),7.38-7.29 (m, 6H), 5.44 (s, 2H), 4.18-4.08 (obscured m, 2H), 3.87-3.75(obscured m, 4H), 3.73-3.65 (obscured m, 2H), 3.63-3.56 (obscured m,2H), 3.53-3.44 (obscured m, 2H), 2.90-2.78 (m, 2H), 2.75-2.55 (obscuredm, 6H), 1.78-1.65 (m, 1H), 1.55 (m, 1H).

LC/MS (System A): m/z (ESI⁺)=579 [MH⁺], R_(t)=0.74 min, UV purity=100%.

Intermediate 104—Synthesis of(2R,3R,4R,5S)-6-[(3-aminopropyl)[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino]hexane-1,2,3,4,5-pentoldihydrochloride

A mixture of(1R,2S)-3-[(3-aminopropyl)[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino]-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol;bis(formic acid), Intermediate 103 (335 mg, 0.50 mmol) and aqueous HClsolution (2 M, 5 ml, 10 mmol) was stirred at RT for 3 h. The reactionmixture was concentrated in vacuo then the residue was azeotroped withMeCN (3×10 ml) to afford the product as a colourless viscous oil (235mg, 99%).

¹H NMR (500 MHz, CD₃OD) δ 4.25-4.18 (m, 2H), 3.91-3.83 (m, 2H),3.81-3.76 (m, 2H), 3.74-3.64 (m, 6H), 3.62-3.51 (m, 2H), 3.50-3.39 (m,4H), 3.10-3.03 (m, 2H), 2.22-2.13 (m, 2H).

LC/MS (System A): m/z (ESI⁺)=403 [MH⁺], R_(t)=0.12 min, ELS purity=100%.

Intermediate 105—Synthesis of tert-butyl4-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)piperidine-1-carboxylate

A solution of 2,5-dioxopyrrolidin-1-yl 9H-fluoren-9-ylmethyl carbonate(5.05 g, 15.0 mmol) in MeCN (50 ml) was added dropwise over 20 min to amixture of tert-butyl 4-aminopiperidine-1-carboxylate (3.00 g, 15.0mmol) and NaHCO₃ (2.52 g, 30.0 mmol) in MeCN (50 ml) and water (50 ml).The resulting mixture was left to stir at RT for 16 h then partitionedbetween EtOAc (100 ml) and water (100 ml). The phases were separatedthen the organic phase was washed with water (100 ml) and brine (100 ml)then dried over Na₂SO₄ and concentrated in vacuo to afford the productas a white foam (6.20 g, 95%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.89 (d, J=7.5 Hz, 2H), 7.69 (d, J=7.4 Hz,2H), 7.41 (t, J=7.4 Hz, 2H), 7.33 (td, J=7.4, 0.9 Hz, 2H), 7.27 (d,J=7.7 Hz, 1H), 4.31 (d, J=6.7 Hz, 2H), 4.21 (t, J=6.6 Hz, 1H), 3.84 (br.d, J=11.7 Hz, 2H), 3.52-3.40 (m, 1H), 2.80 (br. s, 2H), 1.70 (d, J=10.8Hz, 2H), 1.39 (s, 9H), 1.20-1.29 (m, 2H).

LC/MS (System A): m/z (ESI⁺)=445 [(M⁺Na)⁺], R_(t)=1.38 min, UVpurity=97%.

Intermediate 106—Synthesis of (9H-fluoren-9-yl)methylN-(piperidin-4-yl)carbamate hydrochloride

HCl solution in dioxane (4.0 M, 11 ml, 44 mmol) was added to a solutionof tert-butyl4-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)piperidine-1-carboxylate,Intermediate 105 (6.20 g, 14.7 mmol) in MeCN (100 ml). The resultingsolution was stirred at RT for 2 h. The reaction was re-dosed with HClsolution in dioxane (4.0 M, 2.0 ml, 8.0 mmol) then the reaction mixturewas left to stir at RT for a further 1 h. The reaction mixture wasfiltered then the collected solid was washed with MeCN then dried undervacuum to afford the product as a white solid (4.60 g, 87%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.85 (s, 1H), 8.69 (s, 1H), 7.89 (d, J=7.5Hz, 2H), 7.69 (d, J=7.4 Hz, 2H), 7.50 (d, J=7.2 Hz, 1H), 7.42 (t, J=7.4Hz, 2H), 7.36-7.31 (m, 2H), 4.33 (d, J=6.6 Hz, 2H), 4.22 (t, J=6.4 Hz,1H), 3.62-3.54 (m, 1H), 3.21 (d, J=12.0 Hz, 2H), 2.96-2.88 (m, 2H), 1.88(d, J=12.1 Hz, 2H), 1.67-1.50 (m, 2H).

LC/MS (System A): m/z (ESI⁺)=323 [MH⁺], R_(t)=0.89 min, UV purity=100%.

Intermediate 107—Synthesis of (9H-fluoren-9-yl)methylN-{1-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]piperidin-4-yl}carbamate

4,6-O-benzylidene-D-glucopyranose (2.99 g, 11.2 mmol) was added to asolution of (9H-fluoren-9-yl)methyl N-(piperidin-4-yl)carbamatehydrochloride, Intermediate 106 (2.00 g, 5.57 mmol) in MeOH (75 ml). Thereaction mixture was stirred at RT for 20 min then AcOH (670 μl, 11.7mmol) and NaCNBH₃ (700 mg, 11.2 mmol) were added. The reaction mixturewas stirred at RT for 22 h. Saturated aqueous NaHCO₃ solution (50 ml)was added dropwise over 10 min. The resulting mixture was partitionedbetween EtOAc (200 ml) and water (100 ml). The phases were separatedthen the organic phase was washed with saturated aqueous NaHCO₃ solution(100 ml), water (2×100 ml) and brine (2×100 ml). The combined organicphases were dried over Na₂SO₄ and concentrated in vacuo to afford theproduct as a white solid (3.13 g, 92%).

¹H NMR (500 MHz, CD₃OD) δ 7.78 (d, J=7.4 Hz, 2H), 7.63 (d, J=7.1 Hz,2H), 7.52-7.45 (m, 2H), 7.39-7.27 (m, 7H), 5.55 (s, 1H), 4.34 (d, J=6.4Hz, 2H), 4.25 (dd, J=10.7, 5.4 Hz, 1H), 4.21-4.17 (m, 1H), 4.07-4.00 (m,1H), 3.97-3.92 (m, 1H), 3.90-3.87 (d, J=6.4 Hz, 1H), 3.82 (d, J=9.3 Hz,1H), 3.61 (t, J=10.5 Hz, 1H), 3.39-3.32 (m, 1H), 2.97-2.94 (m, 1H),2.77-2.64 (m, 2H), 2.47 (dd, J=11.8, 6.7 Hz, 1H), 2.14 (t, J=10.5 Hz,1H), 2.06-2.02 (m, 1H), 1.75 (dd, J=59.2, 11.6 Hz, 2H), 1.53-1.36 (m,2H).

LC/MS (System A): m/z (ESI⁺)=575 [MH⁺], R_(t)=0.96 min, UV purity=94%.

Intermediate 108—Synthesis of(1R,2S)-3-(4-aminopiperidin-1-yl)-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol

Diethylamine (2.64 ml, 25.6 mmol) was added to a solution of(9H-fluoren-9-yl)methylN-{1-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]piperidin-4-yl}carbamate,Intermediate 107 (94%, 3.13 g, 5.12 mmol) in THF (40 ml). The reactionmixture was left to stir at RT for 1 h. The reaction mixture wasre-dosed with diethylamine (2.64 ml, 25.6 mmol) then the reaction wasleft to stir at RT for a further 20 h. The reaction mixture wasconcentrated in vacuo then the resultant residue was suspended in EtOAc(10 ml) and water (10 ml). Diethyl ether (50 ml) was added then themixture was sonicated. The resulting suspension was filtered then thecollected solid was rinsed with diethyl ether (20 ml) then dried undervacuum to afford the product as a white solid (1.98 g, >99%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.41 (dd, J=6.5, 3.1 Hz, 2H), 7.38-7.31 (m,3H), 5.49 (s, 1H), 4.11 (dd, J=10.5, 4.9 Hz, 1H), 3.81 (q, J=6.2 Hz,1H), 3.79-3.69 (m, 3H), 3.52-3.48 (m, 1H), 2.83 (d, J=11.3 Hz, 1H),2.58-2.52 (m, 1H), 2.45 2.45 (m, 2H+DMSO), 2.24 (dd, J=12.4, 6.1 Hz,1H), 1.93-1.73 (m, 2H), 1.61 (d, J=12.5 Hz, 1H), 1.49 (d, J=12.1 Hz,1H), 1.17-1.14 (m, 2H).

LC/MS (System A): m/z (ESI⁺)=353 [MH⁺], R_(t)=0.13 min, ELS purity=100%.

Intermediate 109—Synthesis of(2R,3R,4R,5S)-6-(4-aminopiperidin-1-yl)hexane-1,2,3,4,5-pentoldihydrochloride

Aqueous HCl solution (2.0 M, 11 ml, 22 mmol) was added to a suspensionof(1R,2S)-3-(4-aminopiperidin-1-yl)-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol,Intermediate 108 (400 mg, 1.13 mmol) in water (5 ml). The reactionmixture was left to stir at RT for 2 h then concentrated in vacuo. Theresulting residue was dissolved in water (20 ml) then extracted withEtOAc (20 ml). The aqueous phase was concentrated in vacuo to afford theproduct as an off-white foam (279 mg, 73%).

¹H NMR (500 MHz, CD₃OD) δ 4.26-4.18 (m, 1H), 3.87-3.74 (m, 4H),3.74-3.62 (m, 4H), 3.61-3.42 (m, 2H), 3.29-3.12 (m, 2H), 2.28 (t, J=13.2Hz, 2H), 2.15-1.92 (m, 2H).

LC/MS (System A): m/z (ESI⁺)=265 [MH⁺], R_(t)=0.13 min, ELS purity=100%.

Intermediate 110—Synthesis of benzyl4-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}piperidine-1-carboxylate

A mixture of benzyl 4-aminopiperidine-1-carboxylate (1.50 g, 6.40 mmol)and 4,6-O-benzylidene-D-glucopyranose (3.44 g, 12.8 mmol) in MeOH (40ml) was stirred at RT for 2 h. AcOH (733 μl, 12.8 mmol) was added,followed by portionwise addition of NaCNBH₃ (805 mg, 12.8 mmol). Thereaction was stirred at RT for 16 h. The reaction was recharged with4,6-O-benzylidene-D-glucopyranose (3.44 g, 12.8 mmol) then the reactionwas stirred at RT for a further 1 h. NaCNBH₃ (805 mg, 12.8 mmol) wasadded then the reaction mixture was stirred at RT for a further 64 h.The reaction was recharged with AcOH (733 μl, 12.8 mmol) then thereaction was stirred at RT for a further 4 h.4,6-O-Benzylidene-D-glucopyranose (3.44 g, 12.8 mmol) was added then thereaction was stirred at RT for 0.5 h. AcOH (733 μl, 12.8 mmol) andNaCNBH₃ (805 mg, 12.8 mmol) were added then the reaction was left tostir at RT for a further 16 h. Saturated aqueous NaHCO₃ solution (20 ml)was added dropwise over 5 min. The resultant mixture was partitionedbetween EtOAc (60 ml) and saturated aqueous NaHCO₃ solution (50 ml). Thephases were separated then the organic phase was washed with saturatedaqueous NaHCO₃ solution (2×50 ml), water (2×50 ml), brine (20 ml), driedover Na₂SO₄, filtered and evaporated to afford a white solid (4.8 g).The resultant solid was dissolved in MeOH:MeCN (1:1, 10 ml) then half ofthe solution was retained crude for future purification whilst the otherhalf was purified by flash column chromatography on C18 (60 g). Thecolumn was eluted with MeCN:water+0.1% NH₄OH using the followinggradient (% MeCN, column volumes): 5%, 2 CV; 5-23%, 4 CV; 23%, 3 CV;23-49%, 5 CV; 49%, 2 CV; 49-100%, 10 CV; 100%, 2 CV. The desiredfractions were combined and concentrated in vacuo to afford a whitesolid (1.39 g). The material thus obtained was further purified by flashcolumn chromatography on C18 (12 g). The column was eluted withMeCN:water+0.1% formic acid using the following gradient (% MeCN, columnvolumes): 5%, 2 CV; 5-32%, 5 CV; 32%, 2 CV; 32-59%, 6 CV; 59-81%, 1 CV,100%, 1 CV. The desired fractions were combined and concentrated invacuo to afford a white solid (1.12 g). The solid thus obtained wasdissolved in EtOAc (50 ml) and extracted with saturated aqueous NaHCO₃solution (2×20 ml) and water (10 ml), then dried over MgSO₄, filteredand evaporated to afford the product as a white solid (0.95 g, 20%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.44-7.24 (m, 15H), 5.49-5.41 (m, 2H),5.15-5.09 (m, 2H), 5.08-4.99 (m, 2H), 4.47-4.43 (m, 2H), 4.41-4.35 (m,2H), 4.17-4.08 (m, 2H), 4.01-3.90 (m, 2H), 3.83-3.74 (m, 2H), 3.75-3.66(m, 4H), 3.64-3.56 (m, 2H), 3.54-3.42 (m, 2H), 2.70-2.33 (m, 7H+DMSO),1.72-1.59 (m, 2H), 1.39-1.27 (m, 1H), 1.16-1.08 (m, 1H).

LC/MS (System A): m/z (ESI⁺)=739 [MH⁺], R_(t)=0.94 min, UV purity=100%.

Intermediate 111—Synthesis of(1R,2S)-3-{[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl](piperidin-4-yl)amino}-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol

A mixture of benzyl4-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}piperidine-1-carboxylate,Intermediate 110 (950 mg, 1.29 mmol) and palladium on carbon (10 wt %137 mg) in EtOH (15 ml) and AcOH (1.5 ml) was stirred under anatmosphere of hydrogen for 18 h at RT. The reaction mixture was filteredthrough a Celite pad then concentrated in vacuo. The residue thusobtained was suspended in MeCN (20 ml) with sonication then concentratedin vacuo. The process was repeated once more with MeCN (20 ml). Theresidue thus obtained was again suspended in MeCN (20 ml) withsonication then allowed to settle. The supernatant was decanted offusing a pipette. The trituration process was repeated with more MeCN(2×20 ml) then the residue was dried under vacuum to afford a whitesolid (770 mg). The solid thus obtained was dissolved in MeOH thenloaded onto a pre-equilibrated SCX cartridge (10 g). The SCX cartridgewas eluted with MeOH then basic components were released by elution witha solution of ammonia in MeOH (7 M). The basic eluent was concentratedin vacuo then the resultant oil was suspended in MeCN (15 ml) andconcentrated in vacuo. The residue was further azeotroped with MeCN(2×15 ml) then dried under vacuum to afford the product as a white solid(595 mg, 77%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.48-7.22 (m, 11H), 5.49-5.45 (m, 2H),5.17-5.06 (m, 2H), 4.52-4.30 (m, 4H), 4.18-4.06 (m, 2H), 3.86-3.76 (m,2H), 3.73-3.66 (m, 4H), 3.64-3.55 (m, 2H), 3.54-3.44 (m, 2H), 2.96-2.77(m, 2H), 2.69-2.60 (m, 2H+DMSO), 2.58-2.44 (m, 1H+DMSO), 2.44-2.35 (m,2H), 2.34-2.26 (m, 1H), 2.21-2.13 (m, 1H), 1.65-1.50 (m, 2H), 1.40-1.27(m, 1H), 1.22-1.06 (m, 1H).

LC/MS (System A): m/z (ESI⁺)=605 [MH⁺], R_(t)=0.75 min, UV purity=100%.

Intermediate 112—Synthesis of(2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl](piperidin-4-yl)amino}hexane-1,2,3,4,5-pentoldihydrochloride

A mixture of(1R,2S)-3-{[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl](piperidin-4-yl)amino}-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol,Intermediate 111 (315 mg, 0.520 mmol) and aqueous HCl solution (2 M, 5ml, 10 mmol) was stirred at RT for 2 h. The reaction mixture wasconcentrated in vacuo then azeotroped with MeCN to afford the product asa white solid (259 mg, 99%).

¹H NMR (500 MHz, D₂O) b 4.35-4.26 (m, 2H), 4.13-3.98 (m, 1H), 3.96-3.41(m, 16H), 3.30-3.14 (m, 2H), 2.56-2.35 (m, 2H), 2.28-2.15 (m, 1H),2.10-2.04 (m, 1H).

LC/MS (System A): m/z (ESI⁺)=429 [MH⁺], R_(t)=0.75 min, ELS purity=100%.

Intermediate 113—Synthesis of benzylN-[1-(2-{[(tert-butoxy)carbonyl]amino}ethyl)piperidin-4-yl]carbamate

Triethylamine (515 μl, 3.69 mmol) was added to a solution of benzylN-(4-piperidyl)carbamate hydrochloride (500 mg, 1.85 mmol) andtert-butyl N-(2-bromoethyl)carbamate (500 mg, 2.22 mmol) in MeCN (4 ml)in a pressure tube. The tube was sealed then the reaction mixture washeated at 85° C. for 16 h. Additional tert-butylN-(2-bromoethyl)carbamate (150 mg, 0.67 mmol) was added then thereaction was left to heat at 85° C. for a further 1 h. The reactionmixture was concentrated in vacuo then the solid thus obtained wasdissolved in the minimum of refluxing MeCN then allowed to cool to RT.The resultant suspension was filtered then the filtrate was concentratedin vacuo. The crude material was purified by flash column chromatographyon a silica column (25 g). The column was eluted with CH₂Cl₂:MeOH,increasing the gradient linearly from 0-15% MeOH over 10 column volumes.The desired fractions were combined and evaporated to afford a viscousred oil (254 mg). The material thus obtained was partitioned betweenEtOAc (15 ml) and saturated aqueous NaHCO₃ solution (15 ml). The phaseswere separated then the organic phase was washed with water (2×15 ml)and brine (15 ml) then dried over Na₂SO₄ and concentrated in vacuo toafford the product as a pale red solid (140 mg, 20%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.39-7.28 (m, 5H), 7.21 (d, J=7.6 Hz, 1H),6.59 (t, J=5.3 Hz, 1H), 5.00 (s, 2H), 3.29-3.22 (m, 1H), 3.00 (q, J=6.4Hz, 2H), 2.76 (d, J=11.5 Hz, 2H), 2.28 (t, J=6.9 Hz, 2H), 1.95 (t,J=11.0 Hz, 2H), 1.69 (d, J=10.5 Hz, 2H), 1.43-1.32 (m, 11H).

LC/MS (System A): m/z (ESI⁺)=378 [MH⁺], R_(t)=0.85 min, UV purity=100%.

Intermediate 114—Synthesis of tert-butylN-[2-(4-aminopiperidin-1-yl)ethyl]carbamate

A mixture of benzylN-[1-(2-{[(tert-butoxy)carbonyl]amino}ethyl)piperidin-4-yl]carbamate,Intermediate 113 (140 mg, 0.370 mmol) and palladium on carbon (10 wt %,20 mg) in EtOH (5 ml) was stirred under a hydrogen atmosphere at RT for2 h. The reaction mixture was filtered through a Celite pad then thefiltrate was concentrated in vacuo to afford the product as a colourlessoil (124 mg, 96%—yield corrected for 70% purity determined by NMR).

¹H NMR (500 MHz, DMSO-d₆) δ 6.61-6.53 (m, 1H), 4.37 (s, 2H), 3.00 (q,J=6.5 Hz, 2H), 2.76-2.71 (m, 2H), 2.49-2.45 (m, 1H), 2.27 (t, J=7.0 Hz,2H), 1.91 (t, J=10.6 Hz, 2H), 1.67-1.60 (m, 2H), 1.37 (s, 9H), 1.22-1.15(m, 2H).

LC/MS (System A): m/z (ESI⁺)=244 [MH⁺], R_(t)=0.14 min, ELS purity=100%.

Intermediate 115—Synthesis of bis(formic acid); tert-butylN-[2-(4-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}piperidin-1-yl)ethyl]carbamate

A mixture of tert-butyl N-[2-(4-aminopiperidin-1-yl)ethyl]carbamate,Intermediate 114 (70%, 725 mg, 2.08 mmol),4,6-O-benzylidene-D-glucopyranose (3.57 g, 12.7 mmol) and AcOH (725 uL,12.7 mmol) in MeOH (20 ml) was stirred at RT for 0.5 h. NaCNBH₃ (795 mg,12.7 mmol) was added then the resulting mixture was stirred at RT for 5days. The reaction was retreated with 4,6-O-benzylidene-D-glucopyranose(1.80 g, 6.29 mmol) and AcOH (362 uL, 6.32 mmol) then the reaction wasleft to stir at RT for 0.5 h. NaCNBH₃ (396 mg, 6.30 mmol) was added thenthe reaction was left to stir at RT for a further 18 h. The reaction wasretreated with 4,6-O-benzylidene-D-glucopyranose (1.80 g, 6.29 mmol) andAcOH (362 uL, 6.32 mmol) then the reaction was left to stir at RT for0.5 h. NaCNBH₃ (396 mg, 6.30 mmol) was added then the reaction was leftto stir at RT for a further 18 h. The reaction mixture was concentratedunder a stream of nitrogen then saturated aqueous NaHCO₃ solution wasadded dropwise until effervescence ceased. The resulting mixture waspartitioned between saturated aqueous NaHCO₃ solution (150 ml) and EtOAc(150 ml). The phases were separated then the organic phase was washedwith NaHCO₃ (150 ml), water (2×150 ml) and brine (150 ml), then driedover Na₂SO₄ and concentrated in vacuo to afford a pale yellow solid. Thecrude material was purified by flash column chromatography on C18 (120g). The column was eluted with MeCN:water+0.1% formic acid using thefollowing gradient (% MeCN, column volumes): 10%, 2 CV; 10-16%, 11 CV;16%-100%, 4 CV; 100%, 1 CV. The desired fractions were combined andconcentrated in vacuo to afford the product as a colourless oil (383 mg,18%).

LC/MS (System A): m/z (ESI⁺)=375 [(M⁺)⁺H+], 748 [MH⁺], R_(t)=0.82 min,UV purity=83%.

Intermediate 116—Synthesis of(2R,3R,4R,5S)-6-{[1-(2-aminoethyl)piperidin-4-yl][(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}hexane-1,2,3,4,5-pentoltrihydrochloride

A mixture of tert-butylN-[2-[4-[bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino]-1-piperidyl]ethyl]carbamate,Intermediate 115 (83%, 360 mg, 0.356 mmol) and aqueous HCl solution (4.0M, 3.6 ml, 14.4 mmol) was stirred at RT for 0.5 h then concentrated invacuo. The residue was dissolved in water:MeCN (9:1, 12 ml) thenlyophilised to afford the product as a white foam (220 mg, 94%).

¹H NMR (500 MHz, Deuterium Oxide) b 4.34-4.26 (m, 2H), 4.16-4.05 (m,1H), 3.95-3.88 (m, 4H), 3.87 (d, J=2.9 Hz, 1H), 3.84 (d, J=3.0 Hz, 1H),3.83-3.78 (m, 2H), 3.73-3.67 (m, 4H), 3.64-3.46 (m, 8H), 3.40-3.31 (m,2H), 2.58 (d, J=14.0 Hz, 1H), 2.51 (d, J=13.7 Hz, 1H), 2.35-2.24 (m,1H), 2.23-2.12 (m, 1H).

LC/MS (System B): m/z (ESI⁺)=472 [MH⁺], R_(t)=0.29 min, ELS purity=88%.

Intermediate 117—Synthesis of (9H-fluoren-9-yl)methyl4-({[(tert-butoxy)carbonyl]amino}methyl)piperidine-1-carboxylate

NaHCO₃ (2.35 g, 28.0 mmol) was added portionwise over 1 min to a stirredsolution of tert-butyl N-(4-piperidylmethyl)carbamate (3.00 g, 14.0mmol) in MeCN (50 ml) and water (50 ml). A solution of(2,5-dioxopyrrolidin-1-yl) 9H-fluoren-9-ylmethyl carbonate (4.72 g, 14.0mmol) in MeCN (50 ml) was added dropwise over 1 h then the reaction wasleft to stir at RT for 18 h. The reaction mixture was partitionedbetween EtOAc (100 ml) and water (100 ml). Brine was added to aidseparation of phases. The phases were separated then the aqueous phasewas extracted with EtOAc (50 ml). The combined organic extracts werewashed with brine (70 ml), dried over MgSO₄, filtered and concentratedin vacuo to afford the product as an off white solid (7.02 g, 96%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.89 (d, J=7.5 Hz, 2H), 7.62 (d, J=7.4 Hz,2H), 7.41 (t, J=7.3 Hz, 2H), 7.33 (td, J=7.4, 0.9 Hz, 2H), 6.85 (t,J=5.8 Hz, 1H), 4.53-4.29 (m, 2H), 4.26 (t, J=6.3 Hz, 1H), 3.97-3.67 (m,2H), 2.78 (t, J=6.1 Hz, 2H), 2.75-2.61 (m, 2H), 1.60-1.44 (m, 3H), 1.38(s, 9H), 0.93-0.73 (m, 2H). 5 wt % residual solvent.

LC/MS (System A): m/z (ESI⁺)=459 [M⁺Na⁺], R_(t)=1.40 min, UV purity=89%.

Overall purity estimate=84%.

Intermediate 118—Synthesis of (9H-fluoren-9-yl)methyl4-(aminomethyl)piperidine-1-carboxylate hydrochloride

HCl solution in dioxane (4.0 M, 12 ml, 48 mmol) was added drop-wise over8 min to a stirred solution of (9H-fluoren-9-yl)methyl4-({[(tert-butoxy)carbonyl]amino}methyl)piperidine-1-carboxylate,Intermediate 117 (7.00 g, 16.0 mmol) in MeCN (100 ml). The resultingsolution was stirred at RT for 17 h then concentrated in vacuo to affordthe product as a white solid (5.56 g, 82%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.04-7.82 (m, 5H), 7.62 (d, J=7.4 Hz, 2H),7.42 (t, J=7.4 Hz, 2H), 7.34 (td, J=7.4, 0.9 Hz, 2H), 4.42-4.32 (m, 2H),4.27 (t, J=6.3 Hz, 1H), 4.04-3.71 (m, 2H), 2.84-2.69 (m, 2H), 2.67 (d,J=6.8 Hz, 2H), 1.80-1.70 (m, 1H), 1.70-1.58 (m, 2H), 1.04-0.87 (m, 2H).7 wt % residual dioxane.

LC/MS (System A): m/z (ESI⁺)=337 [MH⁺], R_(t)=0.86 min, UV purity=95%.

Overall purity estimate=88%.

Intermediate 119—Synthesis of (9H-fluoren-9-yl)methyl4-({bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}methyl)piperidine-1-carboxylate

AcOH (3.11 ml, 54.3 mmol) was added to a solution of(9H-fluoren-9-yl)methyl 4-(aminomethyl)piperidine-1-carboxylatehydrochloride, Intermediate 118 (5.56 g, 14.9 mmol) and4,6-O-benzylidene-D-glucopyranose (14.6 g, 54.3 mmol) in MeOH (100 ml).The reaction was stirred at RT for 40 min then NaCNBH₃ (3.41 g, 54.3mmol) was added in portions over 50 min. The resulting solution wasstirred at RT for 17 h. The reaction mixture was re-treated with4,6-O-benzylidene-D-glucopyranose (7.29 g, 27.2 mmol) and AcOH (1.56 ml,27.2 mmol) then stirred at RT for 30 min. NaCNBH₃ (1.71 g, 27.2 mmol)was added in portions over 1 h. The resulting solution was stirred at RTfor a further 70 h then added onto saturated aqueous NaHCO₃ solution(200 ml) in portions over 30 min. The resultant suspension was stirredat RT for 1 h then filtered. The solid was washed with water (100 ml)then dried in vacuo to afford a white solid (13.8 g). A portion (5.55 g)of the crude material thus obtained was purified by flash columnchromatography on C18 (400 g). The column was eluted with MeCN:H₂O+0.1%NH₄OH using the following gradient (% MeCN, column volumes): 10%, 2 CV;10-57%, 16 CV; 57%, 9 CV; 59-63%, 2 CV; 100%, 3 CV. The desiredfractions were combined and concentrated in vacuo to afford the productas a beige solid (2.99 g, 23%).

¹H NMR (500 MHz, CD₃OD-d₄) δ 7.86-7.75 (m, 2H), 7.65-7.55 (m, 2H),7.53-7.44 (m, 4H), 7.43-7.37 (m, 2H), 7.36-7.27 (m, 8H), 5.51 (m, 2H),4.64-4.36 (m, 5H), 4.27-4.20 (m, 3H), 4.04-3.92 (m, 4H), 3.86 (m, 2H),3.71 (m, 2H), 3.64-3.57 (m, 2H), 2.73-2.58 (m, 3H), 2.54-2.44 (m, 2H),2.36-2.23 (m, 2H), 1.81-1.42 (m, 3H), 0.89-0.58 (m, 2H).

LC/MS (System B): m/z (ESI⁺)=841 [MH⁺], R_(t)=4.78 min, UV purity=95%.

Intermediate 120—Synthesis of(1R,2S)-3-{[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl][(piperidin-4-yl)methyl]amino}-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol;bis(formic acid)

Piperidine (3.30 ml, 33.4 mmol) was added to a stirred solution of(9H-fluoren-9-yl)methyl4-({bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}methyl)piperidine-1-carboxylate,Intermediate 119 (2.81 g, 3.34 mmol) in THF (40 ml). The reactionmixture was stirred at RT for 18 h then concentrated in vacuo. Theresidue was suspended in Et₂O (30 ml) with sonication then the resultantsuspension was filtered. The solid collected was rinsed with Et₂O (20ml) then dried under vacuum to afford a white solid (3.07 g). A sample(1.78 g) of the crude solid was purified by flash column chromatographyon C18 (120 g). The column was eluted with MeCN:water+0.1% formic acidusing the following gradient (% MeCN, column volumes): 10%, 2 CV;10-16%, 1.5 CV; 16%, 2.5 CV; 16-39%, 6 CV; 39-100%, 1.5 CV; 100% 2 CV.The remaining crude solid material was purified by flash columnchromatography on C18 (120 g). The column was eluted withMeCN:water+0.1% formic acid using the following gradient (% MeCN, columnvolumes): 10%, 2 CV; 10-14%, 2 CV; 14%, 2 CV; 14-17%, 1 CV; 17-55%, 7CV; 55-100%, 1 CV; 100% 4 CV. The desired fractions from both columnswere combined and concentrated in vacuo to afford the product as anoff-white solid (1.58 g, 67%).

¹H NMR (500 MHz, CD₃OD) δ 8.33 (s, 2H), 7.53-7.45 (m, 4H), 7.41-7.31 (m,6H), 5.53 (m, 2H), 4.25 (m, 2H), 4.02 (m, 2H), 3.96 (m, 2H), 3.89 (m,2H), 3.74 (m, 2H), 3.62 (m, 2H), 3.30-3.20 (m, 2H), 2.96-2.80 (m, 4H),2.78-2.68 (m, 2H), 2.65-2.58 (m, 2H), 2.09-2.00 (m, 1H), 1.92-1.68 (m,2H), 1.33-1.18 (m, 2H).

LC/MS (System A): m/z (ESI⁺)=619 [MH⁺], R_(t)=0.73 min, UV purity=100%.

Intermediate 121—Synthesis of(2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl][(piperidin-4-yl)methyl]amino}hexane-1,2,3,4,5-pentoldihydrochloride

A solution of(1R,2S)-3-{[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl][(piperidin-4-yl)methyl]amino}-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol;bis(formic acid), Intermediate 120 (1.52 g, 2.14 mmol) in aqueous HClsolution (2 M, 23 ml, 46 mmol) was stirred at RT for 4.5 h. The reactionwas concentrated in vacuo to afford a viscous yellow gum (1.18 g,quantitative based on 93% estimated purity).

¹H NMR (500 MHz, D₂O) b 4.32-4.21 (m, 2H), 3.89-3.81 (m, 4H), 3.81-3.75(m, 2H), 3.72-3.59 (m, 4H), 3.56-3.48 (m, 6H), 3.39 (m, 2H), 3.09 (m,2H), 2.40-2.27 (m, 1H), 2.21-2.13 (m, 1H), 2.06-1.99 (m, 1H), 1.66-1.53(m, 2H).

LC/MS (System C): m/z (ESI⁺)=443 [MH⁺], R_(t)=0.32 min, ELS purity=100%.

Intermediate 122—Synthesis of formic acid; tert-butyl(3R)-3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}pyrrolidine-1-carboxylate

2-Picoline borane complex (0.86 g, 8.05 mmol) was added to a suspensionof tert-butyl (3R)-3-aminopyrrolidine-1-carboxylate (500 mg, 2.68 mmol)and 4,6-O-benzylidene-D-glucopyranose (2.88 g, 10.7 mmol) in MeOH (5ml). The mixture was heated at 60° C. for 17 h. The reaction mixture wasallowed to cool to RT then concentrated in vacuo. The residue waspartitioned between EtOAc (15 ml) and water (15 ml). The phases wereseparated then the organic phase was washed with water (15 ml) and brine(15 ml) then dried over Na₂SO₄ and concentrated in vacuo. The crudematerial was purified by flash column chromatography on C18 (60 g,Ultra). The column was eluted with MeCN:H₂O+0.1% formic acid using thefollowing gradient (% MeCN, column volumes): 10%, 2 CV; 10-40%, 10 CV;40-100%, 2 CV; 100%, 2 CV. The desired fractions were combined andconcentrated in vacuo then the residual aqueous solution was lyophilisedto afford the product as a white solid (1.39 g, 70%).

¹H NMR (500 MHz, CD₃OD-d₄) δ 8.27 (s, 1H), 7.47 (dd, J=7.2, 2.3 Hz, 4H),7.34 (qd, J=4.7, 1.8 Hz, 6H), 5.51 (s, 2H), 4.24 (dd, J=10.6, 5.4 Hz,2H), 4.04 (s, 2H), 3.95 (td, J=9.9, 5.4 Hz, 2H), 3.89 (dd, J=5.3, 2.2Hz, 2H), 3.75 (dd, J=9.4, 2.2 Hz, 2H), 3.69 (d, J=6.2 Hz, 1H), 3.59 (q,J=11.4, 11.0 Hz, 3H), 3.34-3.29 (m, 1H+CD₃OD), 3.24-3.15 (m, 1H), 2.92(d, J=48.5 Hz, 5H), 1.96 (d, J=31.5 Hz, 1H), 1.75 (s, 1H), 1.45 (s,9H).LC/MS (System A): m/z (ESI⁺)=691 [MH⁺], R_(t)=0.93 min, UVpurity=100%.

Intermediate 123—Synthesis of(2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl][(3R)-pyrrolidin-3-yl]amino}hexane-1,2,3,4,5-pentoldihydrochloride

A suspension of formic acid; tert-butyl(3R)-3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}pyrrolidine-1-carboxylate,Intermediate 122 (1.39 g, 1.89 mmol) in aqueous HCl solution (2 M, 30ml, 60 mmol) was stirred at RT for 18 h. The reaction mixture wasconcentrated in vacuo then re-dissolved in water (20 ml) and lyophilisedto afford the product as a cream foam (1.11 g, quantitative based on 83%estimated purity).

¹H NMR (500 MHz, D₂O) δ 4.67 (p, J=8.4 Hz, 1H), 4.33-4.28 (m, 2H),3.99-3.92 (m, 1H), 3.88 (dd, J=5.0, 2.3 Hz, 2H), 3.83 (dd, J=11.8, 3.0Hz, 2H), 3.81-3.76 (m, 2H), 3.75-3.64 (m, 6H), 3.63-3.58 (m, 2H),3.52-3.46 (m, 2H), 3.47-3.39 (m, 1H), 2.75-2.67 (m, 1H), 2.37-2.28 (m,1H).LC/MS (System A): m/z (ESI⁺)=415 [MH⁺], R_(t)=0.13 min, ELSpurity=100%.

Intermediate 124—Synthesis of formic acid; tert-butyl(3S)-3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}pyrrolidine-1-carboxylate

2-Picoline borane complex (861 mg, 8.05 mmol) was added to a suspensionof tert-butyl (3S)-3-aminopyrrolidine-1-carboxylate (500 mg, 2.68 mmol)and 4,6-O-benzylidene-D-glucopyranose (2.88 g, 10.7 mmol) in MeOH (5ml). The resultant mixture was heated at 60° C. for 16 h. The reactionmixture was allowed to cool to RT partitioned between EtOAc (20 ml) andwater (20 ml). The phases were separated then the aqueous phase wasextracted with EtOAc (20 ml). The combined organic phases were washedwith water (20 ml) and 1:1 water:brine (20 ml) then dried over Na₂SO₄and concentrated in vacuo. The crude material was purified by flashcolumn chromatography on C18 (30 g). The column was eluted withMeCN:H₂O+0.1% formic acid using the following gradient (% MeCN, columnvolumes): 10%, 2 CV; 10-40%, 10 CV; 40-100%, 2 CV; 100%, 2 CV. Thedesired fractions were combined and concentrated in vacuo to afford theproduct as a pale yellow foam (1.73 g, 87%).

¹H NMR (500 MHz, CD₃OD) δ 8.25 (s, 1H), 7.54-7.46 (m, 4H), 7.41-7.30 (m,6H), 5.54 (s, 2H), 4.30-4.22 (m, 2H), 4.14-4.07 (m, 2H), 4.01-3.92 (m,2H), 3.91 (dd, J=5.3, 2.2 Hz, 2H), 3.82-3.72 (m, 3H), 3.68-3.53 (m, 3H),3.41-3.33 (m, 1H), 3.25-3.04 (m, 4H), 3.04-2.89 (m, 2H), 2.17-2.07 (m,1H), 1.99-1.83 (m, 1H), 1.56-1.36 (m, 9H).

LC/MS (System A): m/z (ESI⁺)=691 [MH⁺], R_(t)=0.93 min, ELS purity=100%.

Intermediate 125—Synthesis of(2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl][(3S)-pyrrolidin-3-yl]amino}hexane-1,2,3,4,5-pentoldihydrochloride

A suspension of formic acid; tert-butyl(3S)-3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}pyrrolidine-1-carboxylate,Intermediate 124 (1.72 g, 2.33 mmol) in aqueous HCl solution (2 M, 30ml, 60 mmol) and MeOH (1 ml) was stirred at RT for 4 h. The reactionmixture was concentrated in vacuo then re-dissolved in water andlyophilised to afford the product as a cream foam (1.05 g, 92%).

¹H NMR (500 MHz, CD₃OD) δ 4.72 (p, J=8.2 Hz, 1H), 4.30 (s, 2H),3.98-3.36 (m, 18H), 2.76-2.66 (m, 1H), 2.48-2.36 (m, 1H).

LC/MS (System A): m/z (ESI⁺)=415 [MH⁺], R_(t)=0.13 min, ELS purity=100%.

Intermediate 126—Synthesis of formic acid; tert-butylN-[(1r,4r)-4-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}cyclohexyl]carbamate

2-Picoline borane complex (939 mg, 8.78 mmol) was added to a suspensionof tert-butyl N-(4-aminocyclohexyl)carbamate (627 mg, 2.93 mmol) and4,6-O-benzylidene-D-glucopyranose (3.14 g, 11.7 mmol) in MeOH (6 ml).The mixture as heated at 60° C. for 16 h then concentrated in vacuo. Theresidue was partitioned between EtOAc (15 ml) and water (15 ml). Thephases were separated then the organic phase was washed with water (10ml) and brine (10 ml) then dried over Na₂SO₄ and concentrated in vacuo.The crude material was purified by flash column chromatography on C18(30 g). The column was eluted with MeCN:H₂O+0.1% formic acid using thefollowing gradient (% MeCN, column volumes): 10%, 2 CV; 10-40%, 10 CV;40-100%, 2 CV; 100%, 2 CV. The desired fractions were combined andconcentrated in vacuo then the residual aqueous solution was lyophilisedto afford the product as a white solid (914 mg, 41% yield)).

¹H NMR (500 MHz, CD₃OD) δ 8.52 (s, 1H), 7.54-7.48 (m, 4H), 7.44-7.32 (m,6H), 5.56 (s, 2H), 4.31-4.23 (m, 2H), 4.18-4.06 (m, 2H), 4.02-3.92 (m,4H), 3.82-3.75 (m, 2H), 3.64 (t, J=10.5 Hz, 2H), 3.30-3.01 (m, 6H),2.00-1.76 (m, 4H), 1.56-1.39 (m, 10H), 1.36-1.15 (m, 2H), 1.09-0.97 (m,1H).

LC/MS (System C): m/z (ESI⁺)=719 [MH⁺], R_(t)=2.29 min, ELS purity=100%.

Intermediate 127—Synthesis of(2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl][(1r,4r)-4-aminocyclohexyl]amino}hexane-1,2,3,4,5-pentoldihydrochloride

A suspension of formic acid; tert-butylN-[(1r,4r)-4-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}cyclohexyl]carbamate,Intermediate 126 (910 mg, 1.19 mmol) in aqueous HCl solution (2 M, 20ml, 40 mmol) was stirred at RT for 18 h.

The reaction mixture was concentrated in vacuo then the residue wasdissolved in water (20 ml) and lyophilised to afford the product as acream foam (718 mg, quantitative based on 85% estimated purity).

¹H NMR (500 MHz, D₂O) b 4.19-4.09 (m, 2H), 3.81-3.66 (m, 6H), 3.63-3.51(m, 6H), 3.39-3.14 (m, 4H), 2.24-2.13 (m, 3H), 2.11-2.04 (m, 1H),1.87-1.76 (m, 1H), 1.69-1.45 (m, 3H).

LC/MS (System A): m/z (ESI⁺)=443 [MH⁺], R_(t)=0.13 min, ELS purity=100%.

Intermediate 128—Synthesis of (9H-fluoren-9-yl)methylN-[(1s,4s)-4-{[(tert-butoxy)carbonyl]amino}cyclohexyl]carbamate

A solution of 9H-fluoren-9-ylmethyl carbonochloridate (4.04 g, 15.6mmol) in THF (30 ml) was added dropwise over 5 min to a cooled (0° C.)mixture of tert-butyl N-[(1s,4s)-4-aminocyclohexyl]carbamate (3.35 g,15.6 mmol) and aqueous sodium carbonate solution (1 M, 30 ml, 30 mmol)in THF (60 ml). The reaction was allowed to warm to RT then left to stirat RT for 16 h. The reaction mixture was diluted with water (100 ml)then extracted with EtOAc (100 ml). The organic phase was washed withwater (100 ml) and brine (50 ml) then dried over Na₂SO₄ and concentratedin vacuo to afford the product as a pale beige foam (6.71 g, 91%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.92-7.83 (m, 2H), 7.74-7.56 (m, 2H),7.45-7.36 (m, 2H), 7.36-7.28 (m, 2H), 7.17 (d, J=5.4 Hz, 1H), 6.70-6.58(m, 1H), 4.44-4.19 (m, 3H), 3.43-3.33 (m, 2H), 1.65-1.21 (m, 17H).

LC/MS (System A): m/z (ESI⁺)=459 [M⁺Na⁺], R_(t)=1.35 min, UV purity=93%.

Intermediate 129—Synthesis of (9H-fluoren-9-yl)methylN-[(1s,4s)-4-aminocyclohexyl]carbamate hydrochloride

HCl solution in dioxane (4.0 M, 77 ml, 310 mmol) was added to asuspension of (9H-fluoren-9-yl)methylN-[(1s,4s)-4-{[(tert-butoxy)carbonyl]amino}cyclohexyl]carbamate,Intermediate 128 (6.71 g, 15.4 mmol) in dioxane (80 ml). The reactionwas left to stir at RT for 20 h.

The reaction mixture was concentrated in vacuo to afford the product asa white solid (6.04 g, 92%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.95-7.81 (m, 5H), 7.78-7.62 (m, 2H),7.46-7.40 (m, 2H), 7.36-7.32 (m, 2H), 7.28-7.20 (m, 1H), 4.36-4.16 (m,3H), 3.54-3.44 (m, 1H), 3.12-2.99 (m, 1H), 1.86-1.41 (m, 8H). Residualsolvent estimate: 6.8 wt % LC/MS (System A): m/z (ESI⁺)=337 [MH⁺],R_(t)=0.90 min, UV purity=94%.

Intermediate 130—Synthesis of (9H-fluoren-9-yl)methylN-[(1s,4s)-4-{[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl][(2S,3R)-2,3-dihydroxy-3-[(5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}cyclohexyl]carbamate

A solution of (9H-fluoren-9-yl)methylN-[(1s,4s)-4-aminocyclohexyl]carbamate hydrochloride, Intermediate 129(4.70 g, 12.6 mmol) and 4,6-O-benzylidene-D-glucopyranose (10.1 g, 37.8mmol) in MeOH (100 ml) was stirred at RT for 0.5 h. NaCNBH3 (3.17 g,50.4 mmol) was added then the reaction mixture was left to heat at 60°C. for 4.5 h. More 4,6-O-benzylidene-D-glucopyranose (10.1 g, 37.8 mmol)was added then the reaction was left to heat at 60° C. for 20 h. More4,6-O-benzylidene-D-glucopyranose (10.1 g, 37.8 mmol) was added then thereaction was left to heat at 60° C. for 19 h. The reaction was allowedto cool to RT then added to saturated aqueous NaHCO₃ solution (100 ml)and EtOAc (100 ml). The resulting suspension was left to stir at RT for2 h then filtered through a Celite pad. The pad was rinsed through withEtOAc (50 ml) then the filtrate was transferred to a separating funnel.Saturated aqueous NaHCO₃ solution (50 ml) was added then the phases wereseparated. Water (150 ml) was added to the organic phase then theresultant mixture was left to stir at RT for a further 1 h. Theresultant mixture was transferred to a separating funnel then the phaseswere separated. The organic phase was washed with water (150 ml) andbrine (100 ml) then dried over Na₂SO₄ and concentrated in vacuo toafford a golden viscous oil (19.1 g). The crude material was dissolvedin refluxing isopropanol (200 ml). The mixture was stirred under refluxfor 0.5 h then allowed to cool to RT. The solid was collected byfiltration, then rinsed with IPA and dried under vacuum to afford theproduct as a cream solid (7.68 g). A sample of the solid thus obtained(3.0 g) was purified by flash column chromatography on C18 (120 g). Thecolumn was eluted with MeCN:H₂O+0.1% formic acid using the followinggradient (% MeCN, column volumes): 20%, 2 CV; 20-50%, 10 CV; 50-100%, 2CV; 100%, 2 CV. The desired fractions were combined and concentrated invacuo to afford the product as a white solid (1.46 g, 13%)).

¹H NMR (500 MHz, CD₃OD) δ 8.35 (s, 1H), 7.85-7.77 (m, 2H), 7.67-7.58 (m,2H), 7.53-7.43 (m, 4H), 7.41-7.26 (m, 10H), 5.52 (s, 2H), 4.58-4.35 (m,2H), 4.31-4.23 (m, 2H), 4.23-4.13 (m, 3H), 4.00-3.89 (m, 4H), 3.75 (m,2H), 3.71-3.65 (m, 1H), 3.62 (m, 2H), 3.44-3.32 (m, 4H), 1.89-1.26 (m,8H).

LC/MS (System A): m/z (ESI⁺)=841 [MH⁺], R_(t)=1.09 min, UV purity=99%.

Intermediate 131—Synthesis of(1R,2S)-3-{[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl][(1s,4s)-4-aminocyclohexyl]amino}-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol;bis(formic acid)

Piperidine (615 uL, 6.23 mmol) was added to a solution of(9H-fluoren-9-yl)methylN-[(1s,4s)-4-{[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl][(2S,3R)-2,3-dihydroxy-3-[(5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}cyclohexyl]carbamate,Intermediate 130 (1.46 g, 1.74 mmol) in THF (10 ml). The reaction wasleft to stir at RT for 21 h then concentrated in vacuo. The residue wassuspended in MeOH (10 ml) then filtered. The filtrate was concentratedin vacuo then suspended in MeOH (5 ml) and filtered. The filtrate waspurified by flash column chromatography on C18 (60 g). The column waseluted with MeCN:H₂O+0.1% formic acid using the following gradient (%MeCN, column volumes): 10%, 2 CV; 10-32%, 7 CV; 32-40%, 1 CV; 12 CV;40-100%, 2 CV; 100%, 2 CV. The desired fractions were combined thenconcentrated in vacuo. The residual aqueous solution was lyophilised toafford the product as a white solid (388 mg, 31%).

¹H NMR (500 MHz, CD₃OD) δ 8.40 (s, 2H), 7.51-7.43 (m, 4H), 7.40-7.28 (m,6H), 5.51 (s, 2H), 4.29-4.20 (m, 2H), 4.08-4.02 (m, 2H), 4.00-3.92 (m,2H), 3.88 (dd, J=5.0, 2.5 Hz, 2H), 3.72 (dd, J=9.3, 2.5 Hz, 2H), 3.61(t, J=10.5 Hz, 2H), 3.39-3.34 (m, 1H), 3.07-2.89 (m, 5H), 1.97-1.62 (m,8H).

LC/MS (System A): m/z (ESI⁺)=619 [MH⁺], R_(t)=0.76 min, UV purity=100%.

Intermediate 132—Synthesis of(2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl][(1s,4s)-4-aminocyclohexyl]amino}hexane-1,2,3,4,5-pentol

A solution of(1R,2S)-3-{[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl][(1s,4s)-4-aminocyclohexyl]amino}-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol;bis(formic acid), Intermediate 131 (385 mg, 0.622 mmol) in aqueous HClsolution (2 M, 6 ml, 12 mmol) was stirred at RT for 4 h thenconcentrated in vacuo. The resulting oil was dissolved in water (10 ml)then lyophilised to afford a white foam (323 mg). The material thusobtained was dissolved in water/MeOH then loading on to a pre-wetted SCXcartridge (5 g). The cartridge was eluted with MeOH then the product wasreleased by extensive elution with 7 M ammonia solution in MeOH. Theammonia eluent was concentrated in vacuo. The residue was dissolved inwater/MeCN then lyophilised to afford the product as a white solid (212mg, 77%).

¹H NMR (500 MHz, D₂O) δ 4.30-4.20 (m, 2H), 3.90-3.62 (m, 12H), 3.57-3.49(m, 1H), 3.43-3.31 (m, 2H), 2.21-2.09 (m, 3H), 2.06-1.90 (m, 4H),1.85-1.70 (m, 2H).

LC/MS (System A): m/z (ESI⁺)=443 [MH⁺], R_(t)=0.14 min, ELS purity=100%.

Intermediate 133—Synthesis of formic acid; tert-butylN-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propyl)-N-methylcarbamate

alpha-picoline borane (0.861 g, 8.05 mmol) was added to a suspension oftert-butyl N-(3-aminopropyl)-N-methylcarbamate (0.505 g, 2.68 mmol) and4,6-O-benzylidene-D-glucopyranose (2.88 g, 10.7 mmol) in MeOH (5 ml).The mixture was heated at 60° C. for 16 h. The reaction mixture wasallowed to cool to RT then partitioned between EtOAc (20 ml) and water(20 ml). The phases were separated then the aqueous phase was extractedwith EtOAc (20 ml). The combined organic phases were washed with water(20 ml) and 1:1 water:brine (20 ml) then dried over Na₂SO₄ andconcentrated in vacuo. The crude material was purified by flash columnchromatography on C18 (60 g). The column was eluted with MeCN:H₂O+0.1%formic acid using the following gradient (% MeCN, column volumes): 10%,2 CV; 10-40%, 10 CV; 40-100%, 2 CV; 100%, 2 CV. The desired fractionswere combined and concentrated in vacuo to afford the product as a paleyellow foam (1.19 g, 60%).

¹H NMR (500 MHz, CD₃OD) δ 8.40 (s, 1H), 7.57-7.44 (m, 4H), 7.44-7.32 (m,6H), 5.55 (s, 2H), 4.31-4.23 (m, 2H), 4.24-4.15 (m, 2H), 4.00-3.90 (m,4H), 3.79 (dd, J=9.4, 2.2 Hz, 2H), 3.64 (t, J=10.5 Hz, 2H), 3.41-3.31(m, 3H), 3.25-3.08 (m, 4H), 2.76-2.64 (m, 3H), 1.88-1.75 (m, 2H),1.50-1.39 (m, 9H).

LC/MS (System A): m/z (ESI⁺)=693 [MH⁺], R_(t)=0.94 min, ELS purity=100%.

Intermediate 134—Synthesis of(2R,3R,4R,5S)-6-{[3-(methylamino)propyl][(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}hexane-1,2,3,4,5-pentoldihydrochloride

A suspension of formic acid; tert-butylN-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propyl)-N-methylcarbamate,Intermediate 133 (1.18 g, 1.60 mmol) in aqueous HCl solution (2 M, 30ml, 60 mmol) and MeOH (1 ml) was stirred at RT for 4 h then concentratedin vacuo. The residue was dissolved in water (30 ml) then lyophilised toafford the product as a cream foam (770 mg, 99%).

¹H NMR (500 MHz, CD₃OD) δ 4.29-4.20 (m, 2H), 3.96-3.86 (m, 2H),3.83-3.77 (m, 2H), 3.77-3.65 (m, 6H), 3.63-3.39 (m, 6H), 3.18-3.10 (m,2H), 2.77 (s, 3H), 2.28-2.18 (m, 2H).

LC/MS (System A): m/z (ESI⁺)=417 [MH⁺], R_(t)=0.13 min, ELS purity=100%.

Intermediate 135—Synthesis of formic acid; tert-butylN-(2-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}ethyl)carbamate

4,6-O-Benzylidene-D-glucopyranose (10.05 g, 37.45 mmol) was added to asolution of tert-butyl N-(2-aminoethyl)carbamate (1.50 g, 9.36 mmol) inMeOH (50 ml). The reaction was stirred at RT for 15 min before then AcOH(2.14 ml, 37.5 mmol) was added. The reaction was stirred at RT for afurther 15 min then NaCNBH₃ (2.35 g, 37. 5 mmol) was added portionwiseover 5 min. The reaction was stirred at RT for 16 h then saturatedaqueous NaHCO₃ solution (50 ml) was added dropwise over 15 min. Furthersaturated aqueous NaHCO₃ solution (50 ml) was added, followed by EtOAc(50 ml). The reaction was stirred at RT for 15 min then transferred to aseparating funnel. More EtOAc (100 ml) was added then the phases wereseparated. The aqueous phase was extracted with EtOAc (150 ml), then thecombined organic phases were washed with saturated aqueous NaHCO₃solution (4×200 ml) and brine (50 ml), then dried over Na₂SO₄, filteredand concentrated in vacuo. The crude material was purified by flashcolumn chromatography on C18 (400 g, Ultra). The column was eluted withMeCN:H₂O+0.1% formic acid using the following gradient (% MeCN, columnvolumes): 10%, 2 CV; 10-49%, 8 CV; 49-54%, 0.5 CV; 54-100%, 1 CV. Thedesired fractions were combined and concentrated in vacuo then theresidual aqueous solution was lyophilised to afford the product as awhite solid (2.77 g, 42%).

¹H NMR (500 MHz, CD₃OD) δ 8.34 (s, 1H), 7.53-7.43 (m, 4H), 7.40-7.26 (m,6H), 5.52 (s, 2H), 4.25 (dd, J=10.7, 5.4 Hz, 2H), 4.14 (q, J=5.7 Hz,2H), 3.94 (td, J=10.0, 5.4 Hz, 2H), 3.89 (dd, J=5.0, 2.3 Hz, 2H), 3.75(dd, J=9.4, 2.2 Hz, 2H), 3.61 (t, J=10.5 Hz, 2H), 3.26-2.99 (m, 8H),1.42 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=665 [MH⁺], R_(t)=0.94 min, UV purity=100%.

Intermediate 136—Synthesis of(2R,3R,4R,5S)-6-[(2-aminoethyl)[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino]hexane-1,2,3,4,5-pentoldihydrochloride

A suspension of formic acid; tert-butylN-(2-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}ethyl)carbamate,Intermediate 135 (1.50 g, 2.11 mmol) in aqueous HCl (2 M, 21 ml, 42mmol) was stirred at RT for 68 h. The reaction was concentrated invacuo, then re-dissolved in MeCN/water and concentrated in vacuo. Theresidue was re-dissolved in MeCN/water then lyophilised to afford theproduct as a white solid (1.03 g, 99% based on 94% estimated purity).

¹H NMR (500 MHz, D₂O) b 4.30 (dt, J=8.9, 4.7 Hz, 2H), 3.88 (dd, J=4.9,2.2 Hz, 2H), 3.86-3.71 (m, 6H), 3.71-3.66 (m, 4H), 3.61-3.50 (m, 6H).

LC/MS (System A): m/z (ESI⁺)=389 [MH⁺], R_(t)=0.14 min, ELS purity=100%.

Intermediate 137—Synthesis of tert-butylN-[2-(2-{2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethoxy]ethoxy}ethoxy)ethyl]carbamate

NaHCO₃ (0.574 g, 6.84 mmol) was added portionwise over 2 min to astirred solution of tert-butylN-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl]carbamate (1.00 g, 3.42mmol) in MeCN (15 ml) and water (15 ml) RT. A solution of(2,5-dioxopyrrolidin-1-yl) 9H-fluoren-9-ylmethyl carbonate (1.15 g, 3.42mmol) in MeCN (15 ml) was added dropwise over 30 min. The resultingsolution was stirred at RT for 18 h. EtOAc (30 ml) and brine (10 ml)were added then the phases were separated. The aqueous phase wasextracted with EtOAc (20 ml). The combined organic extracts were washedwith brine (45 ml), dried over MgSO4, then concentrated in vacuo toafford the product as a yellow gum (1.61 g, 76% yield).

¹H NMR (500 MHz, DMSO-d₆) δ 7.89 (d, J=7.5 Hz, 2H), 7.72-7.61 (m, 2H),7.41 (t, J=7.4 Hz, 2H), 7.37-7.27 (m, 3H), 6.74 (t, J=5.4 Hz, 1H), 4.29(d, J=6.9 Hz, 2H), 4.21 (t, J=6.9 Hz, 1H), 3.55-3.44 (m, 8H), 3.40 (t,J=6.0 Hz, 2H), 3.36 (t, J=6.1 Hz, 2H), 3.13 (q, J=5.9 Hz, 2H), 3.05 (q,J=6.0 Hz, 2H), 1.36 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=537 [M⁺Na⁺], R_(t)=1.28 min, UV purity=83%.

Intermediate 138—Synthesis of (9H-fluoren-9-yl)methylN-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethyl)carbamate hydrochloride

HCl solution in dioxane (4 M, 2 ml, 8 mmol) was added to a stirredsolution of tert-butylN-[2-(2-{2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethoxy]ethoxy}ethoxy)ethyl]carbamate,Intermediate 137 (83%, 1.61 g, 2.61 mmol) in MeCN (16 ml). The resultingsolution was left to stir at RT for 22 then concentrated in vacuo toafford the product as a yellow gum (1.57 g, quantitative based on 75%estimated purity).

¹H NMR (500 MHz, DMSO-d₆) δ 7.89 (d, J=7.5 Hz, 2H), 7.83 (s, 3H), 7.69(d, J=7.4 Hz, 2H), 7.42 (t, J=7.4 Hz, 2H), 7.37-7.29 (m, 3H), 4.30 (d,J=6.9 Hz, 2H), 4.21 (t, J=6.8 Hz, 1H), 3.60-3.57 (m, 2H), 3.56-3.48 (m,8H), 3.40 (t, J=6.0 Hz, 2H), 3.13 (q, J=5.9 Hz, 2H), 2.98-2.92 (m, 2H).20 wt % residual solvent.

LC/MS (System A): m/z (ESI⁺)=415 [MH⁺], R_(t)=0.89 min, UV purity=92%.

Intermediate 139—Synthesis of (9H-fluoren-9-yl)methylN-[(14S,15R)-12-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]-14,15-dihydroxy-15-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]-3,6,9-trioxa-12-azapentadecan-1-yl]carbamate;formic acid

AcOH (737 μL, 12.9 mmol) was added to a solution of(9H-fluoren-9-yl)methylN-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethyl)carbamate hydrochloride,Intermediate 138 (75%, 1.57 g, 3.22 mmol) and4,6-O-benzylidene-D-glucopyranose (3.46 g, 12.9 mmol) in MeOH (30 ml).The reaction was stirred at RT for 45 min. NaCNBH₃ (809 mg, 12.9 mmol)was added portionwise over 50 min. The resultant solution was stirred atRT for 40 h. The reaction mixture was treated with4,6-O-benzylidene-D-glucopyranose (1.73 g, 6.45 mmol), AcOH (368 μL,6.43 mmol) and MeOH (10 ml). The reaction was stirred at RT for 1 h thenNaCNBH₃ (403 mg, 6.41 mmol) was added portionwise over 20 min. MeOH (10ml) was added then the reaction mixture was stirred at RT for 70 h.Saturated aqueous NaHCO₃ solution (100 ml) was added portionwise over 5min then EtOAc (100 ml) was added. The phases were separated then theaqueous phase was extracted with EtOAc (3×30 ml). The combined organicphases were washed with saturated aqueous NaHCO₃ solution (2×50 ml),brine (50 ml), then dried over MgSO₄, filtered and concentrated in vacuoto afford a brown gum (3.86 g). A portion (2 g) of the crude materialwas purified by flash column chromatography on C18 (120 g, Ultra). Thecolumn was eluted with MeCN:H₂O+0.1% formic acid using the followinggradient (% MeCN, column volumes): 10%, 2 CV; 10-31%, 8 CV; 31%, 4.5 CV;31-35%, 1.5 CV; 35-47%, 1 CV; 47-61%, 1.5 CV; 100% 2 CV. The remainingcrude material was purified by flash column chromatography on C18 (120g, Ultra). The column was eluted with MeCN:H₂O+0.1% formic acid usingthe following gradient (% MeCN, column volumes): 10%, 2 CV; 10-27%, 4CV; 27%, 0.5 CV; 27-68%, 9 CV; 68%, 0.5 CV; 68-78%, 2 CV; 78-100%, 1.5CV; 100% 1 CV. The desired fractions from both columns were combined andconcentrated in vacuo to afford the product as a light brown resin (1.30g, 48%).

¹H NMR (500 MHz, CD₃OD) δ 8.44 (s, 1H), 7.81 (d, J=7.5 Hz, 2H),7.68-7.59 (m, 2H), 7.52-7.44 (m, 4H), 7.44-7.37 (m, 2H), 7.37-7.30 (m,8H), 5.52 (s, 2H), 4.36 (d, J=6.8 Hz, 2H), 4.28-4.15 (m, 5H), 3.98-3.88(m, 4H), 3.75 (dd, J=9.4, 2.3 Hz, 2H), 3.67-3.44 (m, 14H), 3.40-3.33 (m,4H), 3.31-3.21 (m, 4H).LC/MS (System A): m/z (ESI⁺)=919 [MH⁺],R_(t)=1.02 min, UV purity=93%.

Intermediate 140—Synthesis of(14S,15R)-1-amino-12-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]-15-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]-3,6,9-trioxa-12-azapentadecane-14,15-diol;bis(formic acid)

Piperidine (1.34 ml, 13.6 mmol) was added to a stirred solution of(9H-fluoren-9-yl)methylN-[(14S,15R)-12-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]-14,15-dihydroxy-15-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]-3,6,9-trioxa-12-azapentadecan-1-yl]carbamate;formic acid, Intermediate 139 (93%, 1.25 g, 1.20 mmol) in THF (15 ml).The resulting solution was stirred at RT for 6 h then concentrated invacuo. The residue thus obtained was suspended in Et₂O (10 ml) withsonication. The supernatant was decanted off then the process wasrepeated with more Et₂O (10 ml). The residue thus obtained was purifiedby flash column chromatography on C18 (120 g, Ultra). The column waseluted with MeCN:H₂O+0.1% formic acid using the following gradient (%MeCN, column volumes): 10%, 2 CV; 10-13%, 1.5 CV; 13%, 2.5 CV; 13-22%,5.5 CV; 22-60%, 5.5 CV; 60-83%, 2 CV; 100% 2 CV. The desired fractionswere combined and concentrated in vacuo to afford the product as ayellow solid (849 mg, 89%).

¹H NMR (500 MHz, CD₃OD) δ 8.40 (s, 2H), 7.54-7.43 (m, 4H), 7.41-7.28 (m,6H), 5.53 (s, 2H), 4.26 (dd, J=10.7, 5.4 Hz, 2H), 4.20-4.12 (m, 2H),3.97-3.87 (m, 4H), 3.74 (dd, J=9.4, 2.6 Hz, 2H), 3.70-3.54 (m, 14H),3.29-3.19 (m, 3H), 3.16-3.08 (m, 2H), 3.07-2.99 (m, 1H), 2.98-2.86 (m,2H).

LC/MS (System A): m/z (ESI⁺)=697 [MH⁺], R_(t)=0.74 min, UV purity=100%.

Intermediate 141—Synthesis of(14S,15R,16R,17R)-1-amino-12-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]-3,6,9-trioxa-12-azaoctadecane-14,15,16,17,18-pentoldihydrochloride

A solution of(14S,15R)-1-amino-12-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]-15-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]-3,6,9-trioxa-12-azapentadecane-14,15-diol;bis(formic acid), Intermediate 140 (845 mg, 1.07 mmol) in aqueous HClsolution (2 M, 10 ml, 20 mmol) was stirred at RT for 5.5 h thenconcentrated in vacuo. The residue thus obtained was re-dissolved inwater (15 ml) then lyophilised to afford a pale yellow gum (660 mg,quantitative based on 96% estimated purity).

¹H NMR (500 MHz, D₂O) δ 4.30-4.22 (m, 2H), 3.96-3.91 (m, 2H), 3.88-3.82(m, 4H), 3.81-3.63 (m, 17H), 3.59-3.48 (m, 5H), 3.26-3.20 (m, 2H).

LC/MS (System C): m/z (ESI⁺)=521 [MH⁺], R_(t)=0.33 min, ELS purity=100%.

Intermediate 142—Synthesis of tert-butylN-[2-(4-bromophenyl)ethyl]carbamate

Di-tert-butyl dicarbonate (3.93 g, 18.0 mmol) was added to a cooled (0°C.) stirred solution of 2-(4-bromophenyl)ethanamine (3.00 g, 15.0 mmol)in THF (20 ml). The resulting solution was allowed to warm to RT thenstirred at RT for 18 h. The reaction mixture was partitioned betweenEtOAc (30 ml) and saturated aqueous NaHCO₃ solution (50 ml). The phaseswere separated then the aqueous phase was extracted with EtOAc (15 ml).The combined organic phases were washed with brine (50 ml), dried overMgSO₄, then concentrated in vacuo. The crude material was dissolved inthe minimum volume of CH₂Cl₂, pre-adsorbed onto silica, then purified byflash column chromatography on a silica column (25 g). The column waseluted with EtOAc:heptane, using the following gradient (% EtOAc, columnvolumes): 0%, 1 CV; 0-8%, 2.5 CV; 8-12%, 1 CV; 12%, 3.5 CV; 12-27%, 5.5CV; 27-30%, 0.5 CV; 30%, 2 CV; 30-90%, 4 CV; 100% 1 CV. The desiredfractions were combined and concentrated in vacuo to afford the productas a white solid (3.99 g, 88%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.50-7.42 (m, 2H), 7.19-7.11 (m, 2H), 6.86(t, J=5.3 Hz, 1H), 3.12 (q, J=6.6 Hz, 2H), 2.70-2.63 (m, 2H), 1.41-1.26(m, 9H).

LC/MS (System A): R_(t)=1.27 min, UV purity=99%.

Intermediate 143—Synthesis of tert-butylN-{2-[4′-(cyanomethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamate

A mixture of tert-butyl N-[2-(4-bromophenyl)ethyl]carbamate,Intermediate 142 (4.09 g, 13.6 mmol), [4-cyanomethyl)phenyl]boronic acid(2.63 g, 16.4 mmol) and K₂CO₃ (5.65 g, 40.9 mmol) in 1,4-dioxane (105ml) was degassed by bubbling a stream nitrogen through the mixture for 5min. Pd(dppf)Cl₂·CH₂Cl₂ (445 mg, 0.545 mmol) was added and degassing wascontinued for a further 5 min. The reaction mixture was heated at 80° C.for 15 h then at 100° C. for 7 h. The reaction was allowed to cool to RTthen retreated with K₂CO₃ (3.76 g, 27.2 mmol) and degassed for 5 min.Pd(dppf)Cl₂·CH₂Cl₂ (445 mg, 0.545 mmol) was added then the mixture wasdegassed for a further 5 min. The resultant mixture was heated at 100°C. for 24 h then allowed to cool to RT. The reaction was retreated withK₂CO₃ (1.88 g, 13.6 mmol) and [4-cyanomethyl)phenyl]boronic acid (0.88g, 5.5 mmol) then degassed for 10 min. Pd(dppf)Cl₂·CH₂Cl₂ (445 mg, 0.545mmol) was added then the mixture was degassed for a further 5 min. Thereaction was heated at 100° C. for 18 h then allowed to cool to RT. Thereaction mixture was filtered then the collected solids were washed withEtOAc (50 ml). The combined filtrate was concentrated in vacuo. Theresidue was re-dissolved in EtOAc:heptane (1:1) then filtered through asilica pad. The pad was rinsed with EtOAc:heptane (1:1, 200 ml). Thefiltrate was concentrated in vacuo to afford an off-white solid (3.94g). The silica pad was rinsed through further with EtOAc (200 ml) toafford a brown solid (1.68 g). The brown solid from the EtOAc filtratewas pre-adsorbed onto silica, then purified by flash columnchromatography on a silica column (50 g). The column was eluted withEtOAc:heptane, using the following gradient (% EtOAc, column volumes):0%, 1 CV; 0-30%, 11 CV; 30%, 20 CV; 30-45%, 4.5 CV; 45%, 7.5 CV; 45-50%,1 CV; 50%, 15 CV. The desired fractions were combined and concentratedin vacuo to afford an off-white solid (1.00 g, 21%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.67 (d, J=8.2 Hz, 2H), 7.59 (d, J=8.2 Hz,2H), 7.42 (d, J=8.2 Hz, 2H), 7.28 (d, J=8.1 Hz, 2H), 6.90 (t, J=5.5 Hz,1H), 4.07 (s, 2H), 3.17 (q, J=6.5 Hz, 2H), 2.73 (t, J=7.4 Hz, 2H),1.44-1.29 (m, 9H).

LC/MS (System A): R_(t)=1.27 min, UV purity=97%.

Intermediate 144—Synthesis of tert-butylN-{2-[4′-(2-aminoethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamate

A mixture of tert-butylN-{2-[4′-(cyanomethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamate,Intermediate 143 (570 mg, 1.69 mmol), aqueous ammonia solution (35%, 0.5ml) and aqueous Raney nickel slurry (50%, 2 ml) in EtOH (15 ml) and DMF(5 ml) was stirred under a hydrogen atmosphere for 18 h. The reactionmixture was filtered through a Celite pad. The pad was rinsed with EtOH(50 ml) and MeOH (100 ml) then the combined filtrate was concentrated invacuo. The residue was azeotroped with heptane (3×100 ml) then dried invacuo to afford the product as an off-white solid (515 mg, 84%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.60-7.50 (m, 4H), 7.31-7.22 (m, 4H), 6.89(t, J=5.3 Hz, 1H), 3.19-3.13 (m, 2H), 2.78 (t, J=7.2 Hz, 2H), 2.72 (t,J=7.4 Hz, 2H), 2.66 (t, J=7.2 Hz, 2H), 1.43-1.29 (m, 9H).

LC/MS (System A): m/z (ESI⁺)=341 [MH⁺], R_(t)=0.93 min, UV purity=94%.

Intermediate 145—Synthesis of tert-butylN-{2-[4′-(2-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}ethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamate

AcOH (0.33 ml, 5.8 mmol) was added to a solution of tert-butylN-{2-[4′-(2-aminoethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamate,Intermediate 144 (94%, 515 mg, 1.42 mmol) and4,6-O-benzylidene-D-glucopyranose (1.58 g, 5.89 mmol) in MeOH (50 ml).The reaction was left to stir at RT for 50 min then NaCNBH₃ (370 mg,5.89 mmol) was added portionwise over 25 min. The resulting solution wasstirred at RT for 24 h. Further 4,6-O-benzylidene-D-glucopyranose (790mg, 2.94 mmol), AcOH (0.17 ml, 3.0 mmol) and MeOH (50 ml) were addedthen the reaction was left to stir at RT for 40 min. NaCNBH₃ (185 mg,2.94 mmol) was added portionwise over 20 min then the reaction was leftto stir at RT for 68 h. Further 4,6-O-benzylidene-D-glucopyranose (790mg, 2.94 mmol), AcOH (0.17 ml, 3.0 mmol) and MeOH (50 ml) were addedthen the reaction was left to stir at RT for 30 min. NaCNBH₃ (185 mg,2.94 mmol) was added portionwise over 20 min then the reaction was leftto stir at RT for 18 h. Further 4,6-O-benzylidene-D-glucopyranose (790mg, 2.94 mmol) and MeOH (25 ml) were added then the reaction heated at40° C. for 18 h. The reaction mixture was allowed to cool to RT thensaturated aqueous NaHCO₃ solution (40 ml) was added in portions over 15min. The resultant mixture was stirred at RT for 30 min then the solidwas collected by filtration, rinsed with water (10 ml), then dried invacuo. The crude solid material thus obtained was purified by flashcolumn chromatography on C18 (120 g). The column was eluted withMeCN:H₂O+0.1% NH₄OH using the following gradient (% MeCN, columnvolumes): 10%, 2 CV; 10-59%, 10 CV; 59%, 2 CV; 59-100%, 8 CV; 100%, 2CV.

The desired fractions were combined and concentrated in vacuo to affordthe product as a white solid (932 mg, 78%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.57-7.49 (m, 2H), 7.46-7.37 (m, 6H),7.34-7.29 (m, 6H), 7.28-7.23 (m, 2H), 7.09-7.01 (m, 2H), 6.90 (t, J=5.6Hz, 1H), 5.48 (s, 2H), 5.14 (d, J=5.8 Hz, 2H), 4.50-4.40 (m, 4H), 4.12(dd, J=10.5, 5.3 Hz, 2H), 3.87-3.75 (m, 4H), 3.75-3.69 (m, 2H),3.67-3.60 (m, 2H), 3.50 (t, J=10.4 Hz, 2H), 3.21-3.10 (m, 2H), 2.82-2.65(m, 8H), 2.57 (dd, J=12.9, 8.9 Hz, 2H), 1.43-1.29 (m, 9H).

LC/MS (System B): m/z (ESI⁺)=845 [MH⁺], R_(t)=4.80 min, UV purity=100%.

Intermediate 146—Synthesis of(2R,3R,4R,5S)-6-({2-[4′-(2-aminoethyl)-[1,1′-biphenyl]-4-yl]ethyl}[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino)hexane-1,2,3,4,5-pentoldihydrochloride

A suspension of tert-butylN-{2-[4′-(2-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}ethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamate,Intermediate 145 (932 mg, 1.10 mmol) in aqueous HCl solution (2 M, 8.5ml, 17 mmol) was stirred at RT for 24 h then further aqueous HClsolution (2 M, 8.5 ml, 17 mmol) was added. The reaction was left to stirat RT for a further 24 h. The reaction mixture was heated at 40° C. for4 h then concentrated in vacuo. The residue thus obtained was dissolvedin water (15 ml) then lyophilised to afford the product as a white resin(753 mg, quantitative based on 94% estimated purity).

¹H NMR (500 MHz, D₂O) δ 7.77-7.70 (m, 4H), 7.53-7.48 (m, 2H), 7.48-7.43(m, 2H), 4.30-4.19 (m, 2H), 3.87-3.61 (m, 12H), 3.61-3.45 (m, 4H), 3.33(t, J=7.4 Hz, 2H), 3.29-3.16 (m, 2H), 3.07 (t, J=7.4 Hz, 2H).

LC/MS (System A): m/z (ESI⁺)=569 [MH⁺], R_(t)=0.15 min, ELS purity=100%.

Intermediate 147—Synthesis of(2S)-2-{[(benzyloxy)carbonyl]amino}-4-{[(tert-butoxy)carbonyl]amino}butanoicacid

NaHCO₃ (3.13 g, 37.3 mmol) was added to a solution of(2S)-4-amino-2-{[(benzyloxy)carbonyl]amino}butanoic acid (4.70 g, 18.6mmol) in THF (50 ml) and water (50 ml). The reaction was stirred at RTfor 5 min then a solution of di-tert-butyl dicarbonate (4.88 g, 22.4mmol) in THF (50 ml) was added dropwise over 10 min. The reaction wasstirred at RT for 16 h then concentrated in vacuo to remove the majorityof the THF. The residual aqueous solution was acidified to pH 2 bydropwise addition of 2 M aqueous HCl solution then extracted with EtOAc(100 ml then 50 ml). The combined organic phases were washed with water(50 ml) and brine (50 ml), then dried over Na₂SO₄, filtered andconcentrated in vacuo to afford the product as a colourless oil (6.65 g,98%).

¹H NMR (500 MHz, DMSO-d₆) δ 12.60 (s, 1H), 7.57 (d, J=8.0 Hz, 1H),7.41-7.24 (m, 5H), 6.87-6.76 (m, 1H), 5.03 (s, 2H), 4.00-3.92 (m, 1H),3.06-2.90 (m, 2H), 1.92-1.77 (m, 1H), 1.71-1.59 (m, 1H), 1.37 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=375 [M⁺Na⁺], R_(t)=1.06 min, UV purity=97%.

Intermediate 148—Synthesis of tert-butylN-[(3S)-3-{[(benzyloxy)carbonyl]amino}-3-carbamoylpropyl]carbamate

Isobutyl carbonochloridate (2.94 ml, 22.7 mmol) was added dropwise to acooled (0°) solution of(2S)-2-{[(benzyloxy)carbonyl]amino}-4-{[(tert-butoxy)carbonyl]amino}butanoicacid, Intermediate 147 (6.15 g, 17.5 mmol) and N-methylmorpholine (2.88ml, 26.2 mmol) in THF (100 ml). The reaction was allowed to warm to RTthen stirred at RT for 17 h. The solution was cooled to 0° C. thenammonia solution in MeOH (7 M, 12 ml, 84 mmol) was added dropwise over 5min. The reaction mixture was allowed to warm to RT then stirred at RTfor 3 h. The resultant suspension was filtered then the filtrate wasconcentrated in vacuo. The residue was partitioned between EtOAc (200ml) and saturated aqueous NaHCO₃ solution (100 ml). The phases wereseparated then the organic layer was washed with water (50 ml). Theorganic phase was concentrated in vacuo to afford an off white solid.The solid thus obtained was suspended in MeCN (50 ml) then filtered. Thesolid was dried in vacuo to afford the product as a white solid (2.56 g,40%)

¹H NMR (500 MHz, DMSO-d₆) δ 7.42-7.24 (m, 7H), 7.08-6.97 (m, 1H),6.80-6.68 (m, 1H), 5.09-4.92 (m, 2H), 3.98-3.86 (m, 1H), 3.03-2.88 (m,2H), 1.82-1.68 (m, 1H), 1.67-1.54 (m, 1H), 1.44-1.30 (m, 9H).

LC/MS (System A): m/z (ESI⁺)=374 [M⁺Na⁺], R_(t)=1.52 min, UV purity=97%.

Intermediate 149—Synthesis of tert-butylN-[(3S)-3-amino-3-carbamoylpropyl]carbamate

A mixture of tert-butylN-[(3S)-3-{[(benzyloxy)carbonyl]amino}-3-carbamoylpropyl]carbamate,Intermediate 148 (2.06 g, 5.85 mmol) and 10% Pd/C (50% wet, 0.31 g) (50%wet) in THF (12 ml) and EtOH (12 ml) was stirred under an atmosphere ofhydrogen for 18 h. The reaction was filtered through glass fibre filterpaper, then concentrated in vacuo to afford the product as a white solid(1.27 g, quantitative).

¹H NMR (500 MHz, DMSO-d₆) δ 7.26 (s, 1H), 6.91 (s, 1H), 6.83-6.69 (m,1H), 3.11-2.92 (m, 3H), 1.80-1.59 (m, 3H), 1.46-1.32 (m, 10H).

LC/MS (System A): m/z (ESI⁺)=218 [MH*].

Intermediate 150—Synthesis of tert-butylN-[(3S)-3-[(3-{[(benzyloxy)carbonyl]amino}propyl)amino]-3-carbamoylpropyl]carbamate

Sodium triacetoxyborohydride (2.37 g, 11.2 mmol) was added portionwiseover 5 min to a solution of tert-butylN-[(3S)-3-amino-3-carbamoylpropyl]carbamate, Intermediate 149 (1.62 g,7.46 mmol) and benzyl N-(3-oxopropyl)carbamate (1.55 g, 7.46 mmol) inTHF (40 ml). The reaction was stirred at RT for 16 h then water (50 ml)was added. The mixture was extracted with EtOAc (4×50 ml). The combinedorganic phases were washed with saturated aqueous NaHCO₃ solution (2×50ml) and brine (10 ml), then dried over Na₂SO₄, filtered and concentratedin vacuo to afford the product as a colourless oil (2.89 g, 85%).

¹H NMR (500 MHz, CD₃OD) δ 7.39-7.22 (m, 5H), 5.06 (s, 2H), 3.25-3.06 (m,5H), 2.69-2.45 (m, 2H), 1.85-1.61 (m, 4H), 1.42 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=409 [MH⁺], R_(t)=0.87 min, UV purity=90%.

Intermediate 151—Synthesis of tert-butylN-(3-{[(benzyloxy)carbonyl]amino}propyl)-N-[(1S)-3-{[(tert-butoxy)carbonyl]amino}-1-carbamoylpropyl]carbamate

A solution of di-tert-butyl dicarbonate (1.85 g, 8.49 mmol) in THF (10ml) was dropwise over 5 min to a solution of tert-butylN-[(3S)-3-[(3-{[(benzyloxy)carbonyl]amino}propyl)amino]-3-carbamoylpropyl]carbamate,Intermediate 150 (2.89 g, 7.07 mmol)) and triethylamine (1.47 ml, 10.6mmol) in THF (30 ml). The reaction mixture was left to stir at RT for 16h then concentrated in vacuo. The resultant oil was partitioned betweenEtOAc (50 ml) and water (50 ml). The phases were separated then theorganic phase was washed with saturated aqueous NaHCO₃ solution (50 ml)and brine (20 ml), then dried over Na₂SO₄, filtered and concentrated invacuo. The crude material thus obtained was purified by flash columnchromatography on a silica column (50 g). The column was eluted withCH₂Cl₂:MeOH, increasing the gradient linearly from 100:0 to 92:8 over 15CV. The desired fractions were combined and concentrated in vacuo. Thematerial thus obtained was further purified by flash columnchromatography on C18 (120 g). The column was eluted with MeCN:H₂O+0.1%formic acid using the following gradient (% MeCN, column volumes): 5%, 2CV; 5-100%, 20 CV; 100%, 2 CV. The desired fractions were combined andconcentrated in vacuo to afford the product as an orange oil (0.42 g,12%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.42-7.28 (m, 5H), 7.27-6.96 (m, 3H),6.92-6.61 (m, 1H), 5.08-4.93 (m, 2H), 4.43-3.93 (m, 1H), 3.21-2.78 (m,6H), 2.03-1.52 (m, 4H), 1.46-1.30 (m, 18H).

LC/MS (System A): m/z (ESI⁺)=509 [MH⁺], R_(t)=1.16 min, UV purity=95%.

Intermediate 152—Synthesis of tert-butylN-(3-aminopropyl)-N-[(1S)-3-{[(tert-butoxy)carbonyl]amino}-1-carbamoylpropyl]carbamate

A mixture of tert-butyl N-(3-{[(benzyloxy)carbonyl]amino}propyl)-N-[(1S)-3-{[(tert-butoxy)carbonyl]amino}-1-carbamoylpropyl]carbamate,Intermediate 151 (780 mg, 1.47 mmol) and 10% Pd/C (50% wet, 80 mg) inEtOH (20 ml) was stirred under an atmosphere of hydrogen for 40 h at RT.The reaction mixture was filtered through glass fibre filter paper thenconcentrated in vacuo to afford the product as a white foam (580 mg,quantitative based on 95% estimated purity).

¹H NMR (500 MHz, DMSO-d₆) δ 7.19 (s, 1H), 7.01 (s, 1H), 6.89-6.66 (m,1H), 4.40-3.85 (m, 1H), 3.18-2.83 (m, 5H), 2.49-2.37 (m, 2H+solvent),2.05-1.82 (m, 1H), 1.74-1.30 (m, 22H).

LC/MS (System A): m/z (ESI⁺)=375 [MH⁺], R_(t)=0.80 min, UV purity=100%.

Intermediate 153—Synthesis of formic acid; tert-butylN-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propyl)-N-[(1S)-3-{[(tert-butoxy)carbonyl]amino}-1-carbamoylpropyl]carbamate

4,6-O-Benzylidene-D-glucopyranose (1.60 g, 5.96 mmol) was added to asolution of tert-butylN-(3-aminopropyl)-N-[(1S)-3-{[(tert-butoxy)carbonyl]amino}-1-carbamoylpropyl]carbamate,Intermediate 152 (0.558 g, 1.49 mmol) in MeOH (25 ml). The reactionmixture was stirred at RT for 15 min then AcOH (0.341 ml, 5.96 mmol) wasadded. The reaction mixture was stirred at RT for a further 15 min thenNaCNBH₃ (0.375 g, 5.96 mmol) was added portion-wise over 5 min. Thereaction mixture was stirred at RT for 64 h. The reaction was re-treatedwith 4,6-O-benzylidene-D-glucopyranose (1.6 g, 5.96 mmol) and stirredfor a further 24 h at RT. Saturated aqueous sodium bicarbonate solution(25 ml) was added dropwise over 5 min. EtOAc (20 ml) was added then theresultant mixture was left to stir at RT for 15 min. The phases wereseparated then the organic phase was washed with saturated aqueoussodium bicarbonate solution (4×50 ml) and brine (25 ml). The organicphase was dried over Na₂SO₄, filtered and concentrated in vacuo. Thecrude material was purified by flash column chromatography on C18 (120g). The column was eluted with MeCN:H₂O+0.1% formic acid using thefollowing gradient (% MeCN, column volumes): 10%, 2 CV; 10-46%, 8 CV;46-52%, 1 CV; 52%, 2 CV; 52-97, 2 CV. The desired fractions werecombined and concentrated in vacuo to afford the product as a whitesolid (690 mg, 50%).

¹H NMR (500 MHz, CD₃OD) δ 8.39 (s, 1H), 7.54-7.42 (m, 4H), 7.41-7.26 (m,6H), 5.59-5.47 (m, 2H), 4.33-4.13 (m, 4H), 4.02-3.87 (m, 5H), 3.81-3.70(m, 2H), 3.67-3.55 (m, 2H), 3.42-3.34 (m, 6H+solvent), 3.20-2.99 (m,4H), 1.98-1.78 (m, 4H), 1.52-1.33 (m, 18H).

LC/MS (System A): m/z (ESI⁺)=879 [MH⁺], R_(t)=0.95 min, UV purity=100%.

Intermediate 154—Synthesis of(2S)-4-amino-2-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)amino]butanamidetrihydrochloride

A suspension of formic acid; tert-butylN-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propyl)-N-[(1S)-3-{[(tert-butoxy)carbonyl]amino}-1-carbamoylpropyl]carbamate,Intermediate 153 (690 mg, 0.746 mmol) in aqueous HCl solution (2.0 M,7.5 ml, 15 mmol) was stirred at RT for 16 h. The reaction mixture wasconcentrated in vacuo. The residue was dissolved in MeCN/water thenlyophilised to afford the product as a white solid (500 mg, quantitativebased on 91% estimated purity).

¹H NMR (500 MHz, D₂O) b 4.31-4.21 (m, 2H), 4.15-4.08 (m, 1H), 3.91-3.74(m, 6H), 3.73-3.61 (m, 4H), 3.58-3.42 (m, 6H), 3.29-3.10 (m, 4H),2.43-2.20 (m, 4H).

LC/MS (System A): m/z (ESI⁺)=503 [MH⁺], R_(t)=0.13 min, ELS purity=100%.

Intermediate 155—Synthesis of4-[4-(4-aminobutyl)phenyl]-2-[(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propyl)amino]butanamide;tetrakis(acetic acid)

Intermediate 155 was synthesised according to literature procedures(WO2014/099673 A1).

Intermediate 156—Synthesis of4-[4-(4-aminobutyl)phenyl]-2-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)amino]butanamidetrihydrochloride

A solution of4-[4-(4-aminobutyl)phenyl]-2-[(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propyl)amino]butanamide;tetrakis(acetic acid), Intermediate 155 (96%, 140 mg, 0.13 mmol) inaqueous HCl solution (2 M, 5 ml, 10 mmol) was stirred at RT for 2 h. Thereaction mixture was concentrated in vacuo then lyophilised to affordthe product as a beige solid (107 mg, quantitative based on 88%estimated purity).

¹H NMR (500 MHz, CD₃OD) δ 7.22-7.14 (m, 4H), 4.27-4.18 (m, 2H),4.06-3.99 (m, 1H), 3.91-3.85 (m, 2H), 3.83-3.75 (m, 2H), 3.75-3.63 (m,6H), 3.63-3.39 (m, 6H), 3.23-3.08 (m, 2H), 2.96-2.88 (m, 2H), 2.76-2.60(m, 4H), 2.39-2.13 (m, 4H), 1.75-1.62 (m, 4H).

LC/MS (System A): m/z (ESI⁺)=635 [MH⁺], R_(t)=0.16 min, ELS purity=100%.

Intermediate 157—Synthesis of tert-butyl2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylate

N,N-Dimethylformamide di-tert-butyl acetal (3.00 ml, 12.5 mmol) wasadded to a suspension of2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylicacid, Intermediate 78 (1.00 g, 3.13 mmol) in toluene (10 ml). Thereaction was left to heat at 80° C. for 2 h then allowed to cool to RT.N,N-Dimethylformamide di-tert-butyl acetal (3.00 ml, 12.5 mmol) wasadded then the reaction was left to heat at 80° C. for 16 h.N,N-Dimethylformamide di-tert-butyl acetal (3.00 ml, 12.5 mmol) wasadded then the reaction was left to heat at 100° C. for 6 h then allowedto cool to RT. The reaction mixture was diluted with EtOAc (50 ml) thenwashed with water (2×20 ml), saturated aqueous NaHCO₃ solution (2×20 ml)and brine (10 ml) then dried over Na₂SO₄ and concentrated in vacuo toafford an orange solid (1.6 g). The crude material was purified by flashcolumn chromatography on a silica column (25 g). The column was elutedwith EtOAc:heptane, using the following gradient (% EtOAc, columnvolumes): 0%, 3 CV; 0-53%, 10.5 CV; 53%, 4.5 CV; 53-100%, 9.5 CV. Thedesired fractions were combined and evaporated to afford the product asan off-white solid (460 mg, 37%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.08-8.05 (m, 1H), 7.76 (dd, J=8.4, 1.5 Hz,1H), 7.62 (d, J=8.4 Hz, 1H), 7.51 (t, J=5.4 Hz, 1H), 4.46 (d, J=5.8 Hz,2H), 4.33 (q, J=7.2 Hz, 2H), 1.57 (s, 9H), 1.45-1.23 (m, 12H).

LC/MS (System A): m/z (ESI⁺)=376 [MH⁺], R_(t)=1.14 min, UV purity=94%.

Intermediate 158—Synthesis of6-[(tert-butoxy)carbonyl]-2-({[(tert-butoxy)carbonyl]amino}methyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide

Iodoethane (219 μl, 2.67 mmol) was added to a suspension of tert-butyl2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylate,Intermediate 157 (250 mg, 0.670 mmol) in MeCN (2.5 ml). The reaction washeated under microwave irradiation for 2 h at 120° C. In a separatevial, iodoethane (175 μl, 2.14 mmol) was added to a suspension oftert-butyl2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylate,Intermediate 157 (200 mg, 0.532 mmol) in MeCN (2 ml). The resultantmixture was heated under microwave irradiation for 2 h at 120° C. Thetwo reactions were combined then concentrated in vacuo. The crudematerial was purified by flash column chromatography on a silica column(25 g). The column was eluted with MeOH:CH₂Cl₂, increasing the gradientlinearly from 0:100 to 8:92 over 12 CV. The desired fractions werecombined and evaporated to afford a pink foam (550 mg). The materialthus obtained was further purified by flash column chromatography on asilica column (25 g). The column was eluted with MeOH:CH₂Cl₂, increasingthe gradient linearly from 0:100 to 5:95 over 14 CV. The desiredfractions were combined and evaporated to afford the product as a pinkfoam (240 mg, 38%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.54 (s, 1H), 8.18 (s, 2H), 8.01 (t, J=5.2Hz, 1H), 4.81 (d, J=5.3 Hz, 2H), 4.71 (q, J=7.2 Hz, 2H), 4.64 (q, J=7.2Hz, 2H), 1.61 (s, 9H), 1.51-1.18 (m, 15H).

LC/MS (System C): m/z (ESI⁺)=404 [M⁺], R_(t)=2.56 min, UV purity=100%.

Intermediate 159—Synthesis of2-(aminomethyl)-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrochloride chloride

HCl solution in dioxane (4.0 M, 1.1 ml, 4.4 mmol) was added to asolution of6-[(tert-butoxy)carbonyl]-2-({[(tert-butoxy)carbonyl]amino}methyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 158 (465 mg, 0.870 mmol) in MeCN (5 ml). Thereaction was left to stir at RT for 16 h. HCl solution in dioxane (4.0M, 0.20 ml, 0.80 mmol) was added then the reaction was left to stir atRT for 24 h. The reaction mixture was concentrated in vacuo thenazeotroped with MeCN (2×10 ml). The residue was suspended in MeCN (5 ml)then filtered and dried in vacuo to afford the product as a dark brownsolid (335 mg, quantitative based on 84% estimated purity).

¹H NMR (500 MHz, DMSO-d₆) δ 10.24-8.50 (m, 4H), 8.32-8.21 (m, 2H),4.88-4.66 (m, 6H), 1.56-1.41 (m, 6H).

LC/MS (System A): m/z (ESI⁺)=248 [M⁺], R_(t)=0.14 min, ELS purity=100%.

Intermediate 160—Synthesis of methyl3-amino-5-{[2-(trimethylsilyl)ethoxy]methyl}-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate

NaH dispersion in mineral oil (60%, 458 mg, 11.5 mmol) was addedportionwise over a period of 10 min to a cooled (0° C.) mixture ofmethyl 3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate 2(2.00 g, 10.4 mmol) in DMF (30 ml). The resulting mixture was stirred at0° C. for 20 min then a solution of2-(chloromethoxy)ethyl-trimethyl-silane (2.21 ml, 12.5 mmol) in DMF (5ml) was added dropwise over 3 min. The resulting mixture was stirred at0° C. for 15 min then water (100 ml) was added. The mixture wasextracted with EtOAc (100 ml) then the organic phase was washed withwater (2×100 ml) and brine (100 ml) then dried over Na₂SO₄ andconcentrated in vacuo. The crude material was purified by flash columnchromatography on a silica column (100 g). The column was eluted withEtOAc:heptane, using the following gradient (% EtOAc, column volumes):0%, 1 CV; 0-60%, 10 CV; 60-100%, 1.5 CV; 100%, 1 CV. The desiredfractions were combined and evaporated to afford the product as a yellowsolid (1.87 g, 56%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.67 (d, J=3.9 Hz, 1H), 7.24 (s, 2H), 6.53(d, J=3.9 Hz, 1H), 5.42 (s, 2H), 3.85 (s, 3H), 3.52-3.47 (m, 2H),0.85-0.79 (m, 2H), −0.07-−0.11 (m, 9H).

LC/MS (System A): m/z (ESI⁺)=323 [MH⁺], R_(t)=1.21 min, UV purity=100%.

Intermediate 161—Synthesis of methyl3-amino-7-bromo-5-{[2-(trimethylsilyl)ethoxy]methyl}-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate

NBS (0.773 g, 4.34 mmol) was added portionwise over 5 min to a cooled(0° C.) solution of methyl3-amino-5-{[2-(trimethylsilyl)ethoxy]methyl}-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate,Intermediate 160 (1.56 g, 4.82 mmol) in MeCN (30 ml). The resultantmixture was stirred at 0° C. for 50 min. Additional NBS (0.100 g, 0.562mmol) was added then the reaction was left to stir at 0° C. for afurther 90 min. The reaction mixture was partitioned between EtOAc (100ml) and water (100 ml). The phases were separated then the organicphases were washed with water (100 ml) and brine (100 ml), then driedover Na₂SO₄ and concentrated in vacuo. The crude material was purifiedby flash column chromatography on a silica column (100 g). The columnwas eluted with EtOAc:heptane, using the following gradient (% EtOAc,column volumes): 0%, 1 CV; 0-44%, 7 CV. The desired fractions werecombined and evaporated. The material thus obtained was further purifiedby flash column chromatography on C18 (120 g). The column was elutedwith MeCN:H₂O+0.1% formic acid using the following gradient (% MeCN,column volumes): 10%, 2 CV; 10-76%, 18 CV; 76-96%, 2 CV. The desiredfractions were combined and concentrated in vacuo to afford the productas a yellow solid (584 mg, 30%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.91 (s, 1H), 7.42 (s, 2H), 5.40 (s, 2H),3.88 (s, 3H), 3.54-3.48 (m, 2H), 0.86-0.80 (m, 2H), −0.08 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=401 [M(⁷⁹Br)H*], 403 [M(⁸¹Br)H+],R_(t)=1.33 min, UV purity=100%.

Intermediate 162—Synthesis of methyl3-amino-7-bromo-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate

TFA (2.97 ml, 38.9 mmol) was added to a solution of methyl3-amino-7-bromo-5-{[2-(trimethylsilyl)ethoxy]methyl}-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate,Intermediate 161 (520 mg, 1.30 mmol) in CH₂Cl₂ (5 ml). The resultingmixture was stirred at RT for 2.5 h then concentrated in vacuo. Theresultant residue was dissolved in CH₂Cl₂:MeOH:NH₄OH (2:1:0.5, 5 ml)then left to stir at RT for 2 h. The reaction mixture was concentratedin vacuo.

The crude material was purified by flash column chromatography on asilica column (100 g). The column was eluted with CH₂Cl₂:MeOH, using thefollowing gradient (% MeOH, column volumes): 0%, 1 CV; 0-7.5%, 9 CV;7.5-100% 0.5 CV. The desired fractions were combined and evaporated toafford the product as a yellow/orange solid (309 mg, 83%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.87 (s, 1H), 7.72 (s, 1H), 7.24 (s, 2H),3.87 (s, 3H). LC/MS (System A): m/z (ESI⁺)=271 [M(⁷⁹Br)H*], 273[M(⁸¹Br)H+], R_(t)=0.89 min, UV purity=94%.

Intermediate 163—Synthesis of lithium(1+) ion3-amino-7-bromo-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate

Aqueous LiOH solution (1.0 M, 2.7 ml, 2.7 mmol) was added to asuspension of methyl3-amino-7-bromo-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate162 (361 mg, 1.33 mmol) in MeOH (5 ml). The reaction mixture was stirredat 50° C. for 2.5 h then allowed to cool to RT. The resultant suspensionwas filtered. The solid collected was rinsed with water then dried undersuction to afford a yellow solid (195 mg). The filtrate was concentratedin vacuo then the residue was suspended in water (3 ml). The solid wascollected by filtration, combined with the first batch of solid, thendried in vacuo to afford the product as a yellow solid (243 mg, 67%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.45 (s, 1H), 8.35-6.86 (m, 3H).

LC/MS (System A): m/z (ESI⁺)=257 [M(⁷⁹Br)H+], 259 [M(⁸¹Br)H+],R_(t)=0.82 min, UV purity=96%.

Intermediate 164—Synthesis of7-bromo-2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine

A mixture of lithium(1+) ion3-amino-7-bromo-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate163 (243 mg, 0.924 mmol), CDI (300 mg, 1.85 mmol) and imidazolehydrochloride (116 mg, 1.11 mmol) in DMF (3 ml) was stirred at RT for 1h 15 min. Additional CDI (100 mg, 0.617 mmol) was added then thereaction was left to stir at RT for a further 1 h. The reaction mixturewas diluted with water (5 ml) then stirred at RT for 10 min. Theresultant suspension was filtered then the collected solid was washedwith water (2×5 ml), then dried in vacuo to afford the product as ayellow solid (218 mg, 77%).

¹H NMR (500 MHz, DMSO-d₆) δ 12.05 (s, 1H), 8.89-8.82 (m, 1H), 8.03-7.99(m, 1H), 7.86 (s, 1H), 7.58 (s, 2H), 7.13-7.09 (m, 1H).

LC/MS (System A): m/z (ESI⁺)=307 [M(⁷⁹Br)H+], 309 [M(⁸¹Br)H+],R_(t)=0.81 min, ELS purity=100%.

Intermediate 165—Synthesis of methyl3-amino-7-iodo-5-{[2-(trimethylsilyl)ethoxy]methyl}-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate

NaH dispersion in mineral oil (60%, 240 mg, 6.00 mmol) was addedportionwise over 5 min to a cooled (0° C.) solution of methyl3-amino-7-iodo-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate 10,(95%, 1.82 g, 5.44 mmol) in DMF (25 ml). The resultant mixture wasstirred at 0° C. for 20 min then a solution of2-(chloromethoxy)ethyl-trimethyl-silane (1.01 ml, 5.72 mmol) in DMF (8ml) was added dropwise over 5 min. The reaction mixture was stirred at0° C. for 5 min then water (1 ml) was added. The reaction mixture wasconcentrated in vacuo. The crude material was purified by flash columnchromatography on a silica column (340 g). The column was eluted withEtOAc:heptane, using the following gradient (% EtOAc, column volumes):0%, 1 CV; 0-40%, 6.5 CV. The desired fractions were combined andevaporated to afford the product as a yellow solid (1.85 g, 75%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.98 (s, 1H), 7.47 (s, 2H), 5.48 (s, 2H),3.97 (s, 3H), 3.62-3.56 (m, 2H), 0.94-0.88 (m, 2H), 0.00 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=449 [MH⁺], R_(t)=1.37 min, UV purity=99%.

Intermediate 166—Synthesis of methyl3-amino-7-cyano-5-{[2-(trimethylsilyl)ethoxy]methyl}-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate

Degassed dioxane (3 ml) and water (3 ml) were added to a flaskcontaining methyl3-amino-7-iodo-5-{[2-(trimethylsilyl)ethoxy]methyl}-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate,Intermediate 165 (316 mg, 0.705 mmol), potassium ferrocyanide (149 mg,0.353 mmol), Xphos (17 mg, 0.035 mmol), XPhos Pd G3 (30 mg, 0.035 mmol)and KOAc (14 mg, 0.14 mmol). The resulting mixture was heated at 100° C.for 2 h 20 min then allowed to cool to RT. The reaction mixture waspartitioned between water (20 ml) and EtOAc (20 ml) then the phases wereseparated. The organic phase was washed with water (2×20 ml) and brine(20 ml) then dried over Na₂SO₄ and concentrated in vacuo. The crudematerial was purified by flash column chromatography on a silica column(25 g). The column was eluted with EtOAc:heptane, using the followinggradient (% EtOAc, column volumes): 0%, 1 CV; 0-60%, 10 CV; 60-100%, 1.5CV. The desired fractions were combined and evaporated to afford theproduct as a yellow solid (183 mg, 72%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.62 (s, 1H), 7.56 (s, 2H), 5.47 (s, 2H),3.90 (s, 3H), 3.57-3.52 (m, 2H), 0.88-0.79 (m, 2H), −0.08 (s, 9H).

LC/MS (System A): m/z (ESI⁺)=348 [MH⁺], R_(t)=1.25 min, UV purity=97%.

Intermediate 167—Synthesis of tert-butyl4-[bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino]piperidine-1-carboxylate;formic acid

α-Picoline borane complex (0.801 g, 7.49 mmol) was added to a suspensionof tert-butyl 4-aminopiperidine-1-carboxylate (0.500 g, 2.50 mmol) and4,6-O-benzylidene-D-glucopyranose (2.68 g, 9.99 mmol) in MeOH (5 mL).The mixture was heated at 60° C. for 18 h then allowed to cool to RT.The reaction mixture was concentrated in vacuo then the residue waspartitioned between EtOAc (15 ml) and water (15 ml). The phases wereseparated then the organic phase was washed with water (10 ml) and brine(10 ml) then dried over Na2SO4 and concentrated in vacuo.

The crude material was purified by flash column chromatography on C18(30 g). The column was eluted with MeCN:H2O+0.1% formic acid using thefollowing gradient (% MeCN, column volumes): 10%, 2 CVs; 10-40%, 10 CVs;40-100%, 2 CVs; 100%, 2 CVs. The desired fractions were combined andconcentrated in vacuo then the residual aqueous solution was lyophilisedto afford the product as a white solid (1.07 g, 57%).

LC/MS (System B): m/z (ESI⁺)=705 [MH⁺], R_(t)=2.28 min, ELS purity=100%

1H NMR (500 MHz, Methanol-d4) δ 8.42 (s, 1H), 7.54-7.43 (m, 4H),7.40-7.29 (m, 6H), 5.53 (m, 2H), 4.25 (m, 2H), 4.14-3.89 (m, 8H), 3.76(m, 2H), 3.61 (m, 2H), 3.27-2.96 (m, 5H), 2.74-2.34 (m, 2H), 1.86-1.74(m, 2H), 1.60-1.40 (m, 10H), 1.37-1.26 (m, 1H).

Intermediate 112 (method B)—Synthesis of(2R,3R,4R,5S)-6-[[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]-(4-piperidyl)amino]hexane-1,2,3,4,5-pentoldihydrochloride

A suspension of tert-butyl4-[bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino]piperidine-1-carboxylate;formic acid (290 mg, 0.38 mmol) in aqueous HCl (2 M, 10 mL, 20 mmol) wasstirred at RT for 3 h. The reaction mixture was concentrated in vacuo toafford the product as a yellow hygroscopic solid (225 mg). Yield>100%due to residual water. Purity adjusted to 85% to account for residualwater.

LC/MS (System A): m/z (ESI⁺)=429 [MH⁺], R_(t)=0.14 min, ELS purity=100%

1H NMR (500 MHz, Deuterium Oxide) b 4.31-4.20 (m, 2H), 4.09-3.97 (m,1H), 3.92-3.74 (m, 6H), 3.74-3.38 (m, 10H), 3.25-3.13 (m, 2H), 2.53-2.33(m, 2H), 2.24-1.97 (m, 2H).

Intermediate 168—Synthesis of (9H-fluoren-9-yl)methyl4-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}piperidine-1-carboxylate;formic acid

A mixture of 9H-fluoren-9-ylmethyl 4-aminopiperidine-1-carboxylatehydrochloride (7.80 g, 21.7 mmol) and 4,6-O-benzylidene-D-glucopyranose(23.3 g, 86.9 mmol) in MeOH (110 ml) was stirred at RT for 0.5 h.NaCNBH₃ (5.46 g, 86.9 mmol) was added then the reaction was heated at60° C. The reaction was stirred at 60° C. for 18 h. The reaction wasrecharged with 4,6-O-benzylidene-D-glucopyranose (23.3 g, 86.9 mmol)then left to stir at 60° C. for a further 6 h. The reaction was allowedto cool to RT then added to saturated aqueous NaHCO₃ solution (200 ml)and EtOAc (200 ml). The resultant mixture was filtered through a Celitepad then the filtrate was transferred to a separating funnel. The phaseswere separated then the organic phase was washed with brine:water (1:1,2×200 ml), brine (100 ml), dried over Na₂SO₄, filtered and concentratedin vacuo. The residue was suspended in MeCN (200 ml) and tBME (250 ml)the filtered. The solid obtained was suspended in MeOH then filtered.The combined filtrates were concentrated in vacuo. The crude materialthus obtained was purified by flash column chromatography on C18 (400g). The column was eluted with MeCN:water+0.1% formic acid using thefollowing gradient (% MeCN, column volumes): 20%, 1 CV; 20-50%, 10 CV;50-100%, 2 CV; 100%, 2 CV. The desired fractions were combined andconcentrated in vacuo to remove most of the MeCN and some of the waterthen the residual aqueous solution was lyophilised to afford the productas an off-white solid (12.6 g, 66%).

¹H NMR (500 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.90 (d, J=7.4 Hz, 2H),7.64-7.54 (m, 2H), 7.45-7.27 (m, 14H), 5.46 (s, 2H), 5.23-5.07 (m, 2H),4.88-4.21 (m, 6H), 4.13 (dd, J=10.5, 5.3 Hz, 2H), 4.02-3.67 (m, 9H),3.61 (d, J=9.2 Hz, 2H), 3.50 (t, J=10.5 Hz, 2H), 2.64-2.56 (m, 3H),2.42-2.31 (m, 2H), 1.69-1.51 (m, 2H), 1.31-0.90 (m, 2H).

LC/MS (System A): m/z (ESI⁺)=827 [MH⁺], R_(t)=1.08 min, UV purity=100%.

Intermediate 111 (Method B)—Synthesis of(1R,2S)-3-{[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl](piperidin-4-yl)amino}-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol

Piperidine (9.01 ml, 91.2 mmol) was added to a solution of(9H-fluoren-9-yl)methyl4-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}piperidine-1-carboxylate;formic acid, Intermediate 112 (12.6 g, 14.4 mmol) in THF (150 ml). Thereaction was left to stir at RT for 16 h then concentrated in vacuo. Thecrude solid material was suspended in MeOH (100 ml) then heated todissolve. The solution was allowed to cool then concentrated in vacuountil solid was observed. The resultant suspension was stirred at RT for15 min then filtered. The filtrate was concentrated in vacuo until solidwas observed. The resultant suspension was stirred at RT for 15 min thenfiltered. The filtrate was purified by flash column chromatography onC18 (400 g). The column was eluted with MeCN:H₂O+0.1% formic acid usingthe following gradient (% MeCN, column volumes): 10%, 1 CVs; 10-25%, 6CVs; 25%, 2 CVs; 25-50%, 1 CV; 50-100%, 1 CVs; 100%, 2 CVs. The desiredfractions were combined and concentrated in vacuo to remove the majorityof the solvent. The residual solution thus obtained was lyophilised toafford a pale-yellow solid (6.35 g). The solid thus obtained waspartitioned between EtOAc (100 ml) and saturated aqueous NaHCO₃ solution(100 ml). The phases were separated then the aqueous phase was extractedwith CHCl₃:IPA (2:1, 100 ml) and n-BuOH (2×100 ml). The combined organicphases were dried over Na₂SO₄ then concentrated in vacuo. The residuewas dissolved in 1:2 MeCN:water then lyophilised to afford the productas a white solid (5.81 g, 67%).

¹H NMR (500 MHz, CD₃OD) δ 7.48 (dd, J=7.5, 2.0 Hz, 4H), 7.38-7.28 (m,6H), 5.50 (s, 2H), 4.23 (dd, J=10.6, 5.4 Hz, 2H), 4.00-3.88 (m, 4H),3.85 (dd, J=5.5, 2.4 Hz, 2H), 3.70 (dd, J=9.3, 2.4 Hz, 2H), 3.60 (t,J=10.5 Hz, 2H), 3.09-3.02 (m, 1H), 3.00-2.91 (m, 1H), 2.78 (dd, J=13.4,3.7 Hz, 2H), 2.75-2.65 (m, 1H), 2.59 (dd, J=13.4, 8.8 Hz, 2H), 2.54-2.47(m, 1H), 2.37-2.28 (m, 1H), 1.81-1.70 (m, 2H), 1.55-1.49 (m, 1H),1.42-1.35 (m, 1H).

LC/MS (System A): m/z (ESI⁺)=605 [MH⁺], R_(t)=0.77 min, UV purity=100%.

SYNTHESIS OF EXAMPLE COMPOUNDS Example 1—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-3-methyl-1H-1,3-benzodiazol-3-iumformate

Iodomethane (26 μl, 0.42 mmol) was added to a solution of3-amino-N-[(1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-5H-pyrrolo[2,3-b]pyrazine-2-carboxamide,Intermediate 84 (79%, 35 mg, 0.082 mmol) in DMSO (1 ml). The resultingmixture was stirred at RT for 48 h. Additional iodomethane (30 μl, 0.48mmol) was added and the reaction was stirred at RT for a further 48 h.Additional iodomethane (40 μl, 0.64 mmol) was added and the reaction wasstirred at RT for a further 72 h. The reaction mixture was diluted withMeCN to a final volume of 1.5 ml then purified by preparative HPLC(Method A). The desired fractions were combined and concentrated invacuo to afford the product as a yellow solid (12 mg, 36%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.59 (s, 1H), 9.68 (t, J=5.3 Hz, 1H), 8.47(s, 1H), 8.10-7.98 (m, 2H), 7.74-7.65 (m, 2H), 7.51 (d, J=3.6 Hz, 1H),7.19 (s, 2H), 6.41 (d, J=3.7 Hz, 1H), 5.05 (d, J=5.4 Hz, 2H), 4.69 (q,J=7.2 Hz, 2H), 4.16 (s, 3H), 1.39 (t, J=7.2 Hz, 3H).

LC/MS (System C): m/z (ESI⁺)=350 [M⁺], Rt=1.46 min, UV purity=98%.

Example 2—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-6-fluoro-3-methyl-1H-1,3-benzodiazol-3-iumchloride

A solution of2-(aminomethyl)-1-ethyl-6-fluoro-3-methyl-1H-1,3-benzodiazol-3-iumhydrochloride iodide, Intermediate 19 (182 mg, 0.489 mmol) and DIPEA(170 μl, 0.98 mmol)) in DMF (1.5 ml) was added to a solution oflithium(1⁺) ion 3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate,Intermediate 3 (90 mg, 0.49 mmol), HBTU (204 mg, 0.538 mmol) and DIPEA(170 μl, 0.98 mmol) in DMF (0.5 ml). The reaction was stirred at RT for72 h then concentrated in vacuo. The crude material thus obtained waspurified by flash column chromatography on C18 (12 g). The column waseluted with MeCN:water+0.1% formic acid using the following gradient (%MeCN, column volumes): 5%, 2 CV; 5-69%, 13 CV; 69-100%, 2 CV; 100%, 1CV. The desired fractions were combined and concentrated in vacuo toafford a green/brown solid (30 mg). The solid thus obtained wasdissolved in 2:1 DMSO:MeCN then purified by preparative HPLC (Method A).The desired fractions were combined and concentrated in vacuo to affordthe product as a yellow solid (11 mg, 6%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.51 (s, 1H), 9.66 (t, J=5.3 Hz, 1H),8.12-8.07 (m, 2H), 7.62 (td, J=9.3, 2.4 Hz, 1H), 7.51 (dd, J=3.7, 1.7Hz, 1H), 7.20 (s, 2H), 6.41 (d, J=3.6 Hz, 1H), 5.02 (d, J=5.4 Hz, 2H),4.65 (q, J=7.2 Hz, 2H), 4.16 (s, 3H), 1.37 (t, J=7.2 Hz, 3H).

LC/MS (System C): m/z (ESI⁺)=368 [M⁺], Rt=1.48 min, UV purity=99%.

Example 3—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-chloro-1-ethyl-3-methyl-1H-1,3-benzodiazol-3-iumformate

A suspension of lithium(1⁺) ion3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate 3 (90 mg,0.49 mmol),2-(aminomethyl)-6-chloro-1-ethyl-3-methyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 22 (172 g, 0.489 mmol), EDC·HCl (187 mg, 0.978mmol) and HOAt (66 mg, 0.49 mmol) in DMF (2 ml) was left to stir at RTfor 16 h then concentrated in vacuo. The crude material was purified byflash column chromatography on C18 (12 g). The column was eluted withMeCN:water+0.1% formic acid using the following gradient (% MeCN, columnvolumes): 5%, 6 CV; 10-100%, 20 CV; 100%, 2 CV. The desired fractionswere combined and concentrated in vacuo to afford the product as ayellow solid (38 mg, 18%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.65 (s, 1H), 9.67 (t, J=5.3 Hz, 1H), 8.38(s, 1H), 8.32 (d, J=1.8 Hz, 1H), 8.08 (d, J=8.9 Hz, 1H), 7.76 (dd,J=8.9, 1.8 Hz, 1H), 7.51 (dd, J=3.6, 1.8 Hz, 1H), 7.19 (s, 2H), 6.40 (d,J=3.5 Hz, 1H), 5.03 (d, J=5.3 Hz, 2H), 4.68 (q, J=7.2 Hz, 2H), 4.15 (s,3H), 1.37 (t, J=7.2 Hz, 3H).

LC/MS (System C): m/z (ESI⁺)=384 [M(³⁵Cl)⁺], 386 [M(³⁷Cl)⁺], Rt=1.69min, UV purity=100%.

Example 4—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-6-methoxy-3-methyl-1H-1,3-benzodiazol-3-iumformate

A suspension of2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 4 (230 mg, 1.01 mmol) and2-(aminomethyl)-1-ethyl-6-methoxy-3-methyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 25 (350 mg, 1.01 mmol) in DMF (3 ml) was stirred atRT for 16 h. The resultant suspension was filtered under vacuum. Thesolid thus obtained was washed with MeCN. The combined filtrate wasconcentrated in vacuo then the crude material was purified by flashcolumn chromatography on C18 (30 g). The column was eluted withMeCN:water+0.1% formic acid using the following gradient (% MeCN, columnvolumes): 5%, 2 CV; 5-20%, 5 CV; 20% 1 CV; 20-25%, 2 CV; 25-39%, 1 CV;39-100%, 1 CV; 100%, 1 CV. The desired fractions were combined andconcentrated in vacuo to afford the product as a yellow powder (173 mg,40%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.56 (s, 1H), 9.65 (t, J=5.3 Hz, 1H), 8.35(s, 1H), 7.92 (d, J=9.1 Hz, 1H), 7.56 (d, J=2.2 Hz, 1H), 7.50 (dd,J=3.7, 2.4 Hz, 1H), 7.32-7.26 (m, 1H), 7.20 (s, 2H), 6.44-6.36 (m, 1H),5.00 (d, J=5.4 Hz, 2H), 4.64 (q, J=7.1 Hz, 2H), 4.12 (s, 3H), 3.90 (s,3H), 1.37 (t, J=7.2 Hz, 3H).

LC/MS (System C): m/z (ESI⁺)=380 [M⁺], Rt=1.61 min, UV purity=100%.

Example 5—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-3-methyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-iumformate

A solution of2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 4 (64 mg, 0.28 mmol) and2-(aminomethyl)-1-ethyl-3-methyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-iumhydrochloride iodide, Intermediate 28 (94%, 120 mg, 0.27 mmol) in DMF (1ml) was stirred at RT for 16 h. The reaction mixture was concentrated invacuo then purified by flash column chromatography on C18 (12 g). Thecolumn was eluted with MeCN:water+0.1% formic acid using the followinggradient (% MeCN, column volumes): 5%, 2 CV; 5-24%, 4 CV; 24%, 3 CV;24-52%, 6 CV; 52-100%, 4 CV; 100% 3 CV. The desired fractions werecombined and concentrated in vacuo to afford the product as an orangesolid (60 mg, 48%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.65 (s, 1H), 9.68 (t, J=5.3 Hz, 1H), 8.62(s, 1H), 8.41 (s, 1H), 8.28 (d, J=8.8 Hz, 1H), 8.06 (dd, J=8.8, 1.1 Hz,1H), 7.51 (dd, J=3.7, 1.8 Hz, 1H), 7.19 (s, 2H), 6.40 (d, J=3.6 Hz, 1H),5.08 (d, J=5.3 Hz, 2H), 4.79 (q, J=7.1 Hz, 2H), 4.20 (s, 3H), 1.39 (t,J=7.2 Hz, 3H).

LC/MS (System C): m/z (ESI⁺)=418 [M⁺], Rt=1.84 min, UV purity=100%.

Example 6—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-3-methyl-6-(trifluoromethoxy)-1H-1,3-benzodiazol-3-iumformate

A solution of2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 4 (55 mg, 0.24 mmol) and2-(aminomethyl)-1-ethyl-3-methyl-6-(trifluoromethoxy)-1H-1,3-benzodiazol-3-iumhydrochloride iodide, Intermediate 32 (87%, 105 mg, 0.209 mmol) in DMF(1 ml) was stirred at RT for 16 h. The reaction mixture was concentratedin vacuo then purified by flash column chromatography on C18 (12 g). Thecolumn was eluted with MeCN:water+0.1% formic acid using the followinggradient (% MeCN, column volumes): 5%, 2 CV; 5-28%, 5 CV; 28%, 2 CV;28-43% 3 CV; 43-92%, 2 CV; 100% 3 CV. The desired fractions werecombined and concentrated in vacuo. The residue was suspended in MeCN (5ml) then filtered. The filtrate was concentrated in vacuo then purifiedby flash column chromatography on C18 (12 g). The column was eluted withMeCN:water+0.1% formic acid using the following gradient (% MeCN, columnvolumes): 5%, 2 CV; 5-9%, 1 CV; 9% 1 CV; 9-23%, 3 CV; 23%, 4 CV; 23-32%1 CV; 32-100%, 2 CV, 100% 2 CV. The desired fractions were combined andconcentrated in vacuo to afford the product as a brown solid (38 mg,38%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.77 (s, 1H), 9.69 (t, J=5.3 Hz, 1H), 8.50(s, 1H), 8.34-8.26 (m, 1H), 8.19 (d, J=9.1 Hz, 1H), 7.79-7.70 (m, 1H),7.51 (d, J=3.8 Hz, 1H), 7.20 (s, 2H), 6.40 (d, J=3.8 Hz, 1H), 5.05 (d,J=5.3 Hz, 2H), 4.72 (q, J=7.2 Hz, 2H), 4.18 (s, 3H), 1.38 (t, J=7.2 Hz,3H).

LC/MS (System C): m/z (ESI⁺)=434 [M⁺], Rt=1.97 min, UV purity=100%.

Example 7—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-methoxy-1H-1,3-benzodiazol-3-iumformate

A suspension of lithium(1⁺) ion3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate 3 (40 mg,0.22 mmol),2-(aminomethyl)-1,3-diethyl-6-methoxy-1H-1,3-benzodiazol-3-ium iodide,Intermediate 36 (92%, 78 mg, 0.20 mmol), EDC·HCl (83 mg, 0.43 mmol) andHOAt (30 mg, 0.22 mmol) in DMF (1 ml) was stirred at RT for 72 h thenconcentrated in vacuo. The crude material was purified by flash columnchromatography on C18 (12 g). The column was eluted with MeCN:water+0.1%formic acid using the following gradient (% MeCN, column volumes): 5%, 6CV; 10-100%, 20 CV; 100%, 2 CV. The desired fractions were combined andconcentrated in vacuo to afford the product as a green solid (52 mg,58%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.65 (s, 1H), 9.69 (t, J=5.3 Hz, 1H), 8.40(s, 1H), 7.99-7.90 (m, 1H), 7.59-7.54 (m, 1H), 7.52-7.48 (m, 1H),7.32-7.12 (m, 3H), 6.40 (d, J=3.2 Hz, 1H), 5.08-4.98 (m, 2H), 4.65 (q,J=7.1 Hz, 4H), 3.90 (s, 3H), 1.39 (td, J=7.1, 1.6 Hz, 6H).

LC/MS (System C): m/z (ESI⁺)=394 [M⁺], Rt=1.75 min, UV purity=98%.

Example 8—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-3-benzyl-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-iumformate

A suspension of lithium(1⁺) ion3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate 3 (90 mg,0.49 mmol),2-(aminomethyl)-3-benzyl-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-iumbromide, Intermediate 38 (56%, 260 mg, 0.39 mmol), EDC·HCl (190 mg, 0.98mmol) and HOAt (67 mg, 0.49 mmol) in DMF (2 ml) was stirred at RT for 5h. DMF (2 ml) was added then the reaction was stirred for a further 16h, then concentrated in vacuo. The crude material was dissolved in 2:1DMSO:MeCN then purified by preparative HPLC (Method A). The desiredfractions were combined and concentrated in vacuo to afford the productas a yellow solid (91 mg, 47%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.55 (s, 1H), 9.59 (t, J=5.4 Hz, 1H), 8.44(s, 1H), 7.64 (d, J=9.2 Hz, 1H), 7.61 (d, J=2.3 Hz, 1H), 7.49 (dd,J=3.7, 2.3 Hz, 1H), 7.30-7.04 (m, 8H), 6.36 (dd, J=3.7, 1.3 Hz, 1H),5.91 (s, 2H), 5.13 (d, J=5.4 Hz, 2H), 4.72 (q, J=7.2 Hz, 2H), 3.90 (s,3H), 1.44 (t, J=7.2 Hz, 3H).

LC/MS (System C): m/z (ESI⁺)=456 [M⁺], Rt=2.13 min, UV purity=100%.

Example 9—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-6-methoxy-3-(2-methoxy-2-oxoethyl)-1H-1,3-benzodiazol-3-iumbromide

2-(Aminomethyl)-1-ethyl-6-methoxy-3-(2-methoxy-2-oxoethyl)-1H-1,3-benzodiazol-3-iumhydrochloride bromide, Intermediate 41 (65%, 165 mg, 0.272 mmol) wasadded to a solution of lithium(1⁺) ion3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate 3 (50 mg,0.27 mmol), DIPEA (189 μl, 1.09 mmol) and HBTU (134 mg, 0.353 mmol) inDMF (1 ml). The resulting mixture was stirred at RT for 1.5 h thenconcentrated in vacuo. The crude material was purified by flash columnchromatography on C18 (12 g). The column was eluted with MeCN:water+0.1%formic acid using the following gradient (% MeCN, column volumes): 10%,2 CV; 10-21%, 7 CV; 21%, 1 CV; 21-31%, 7 CV; 31-100%, 6 CV; 100%, 2 CV.The desired fractions were combined and lyophilised to afford a brownsolid (16 mg). The solid thus obtained was dissolved in 2:1 CH₂Cl₂/MeOH(5 ml) then stirred with Dowex 1×2 chloride form for 5 min. The resinwas removed by filtration then rinsed with 2:1 CH₂Cl₂:MeOH. The filtratewas concentrated in vacuo to afford the product as a yellow/orange solid(10 mg, 7%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.52 (s, 1H), 9.56 (t, J=5.5 Hz, 1H), 7.89(d, J=9.2 Hz, 1H), 7.63 (d, J=2.3 Hz, 1H), 7.51 (dd, J=3.7, 2.5 Hz, 1H),7.27 (dd, J=9.1, 2.3 Hz, 1H), 7.17 (s, 2H), 6.41 (dd, J=3.7, 1.6 Hz,1H), 5.65 (s, 2H), 5.03 (d, J=5.5 Hz, 2H), 4.77 (q, J=7.2 Hz, 2H), 3.92(s, 3H), 3.37 (s, 3H), 1.47 (t, J=7.2 Hz, 3H).

LC/MS (System C): m/z (ESI⁺)=438 [M⁺], Rt=1.69 min, UV purity=98%.

Example 10—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-3-(carboxylatomethyl)-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-ium

1.0 M aqueous LiOH solution (86 μl, 0.086 mmol) was added to a solutionof2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-6-methoxy-3-(2-methoxy-2-oxoethyl)-1H-1,3-benzodiazol-3-iumbromide, Example 9 (32 mg, 0.062 mmol) in MeOH (0.5 ml). The resultingsolution was stirred at RT for 15 min. The reaction mixture wasconcentrated under a stream of nitrogen. The residue was suspended in1:1 DMSO:MeCN then filtered through a syringe filter. The filter wasthen washed extensively with MeOH. The combined MeOH washings wereconcentrated to an orange oil. The crude material thus obtained waspurified by flash column chromatography on C18 (12 g). The column waseluted with MeCN:water+0.1% formic acid using the following gradient (%MeCN, column volumes): 10%, 2 CV; 10-23%, 6 CV; 23%, 1 CV; 23-29%, 3 CV;29-100%, 5 CV; 100%, 3 CV. The desired fractions were combined andlyophilised to afford the product as a pale yellow solid (3 mg, 12%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.47 (s, 1H), 9.53 (t, J=5.7 Hz, 1H), 7.91(d, J=9.2 Hz, 1H), 7.52 (d, J=2.3 Hz, 1H), 7.48 (dd, J=3.8, 2.1 Hz, 1H),7.32-7.16 (m, 3H), 6.36 (dd, J=3.8, 1.5 Hz, 1H), 4.97-4.91 (m, 4H), 4.60(d, J=7.4 Hz, 2H), 3.89 (s, 3H), 1.33 (t, J=7.1 Hz, 3H).

LC/MS (System C): m/z (ESI⁺)=424 [MH⁺], Rt=1.56 min, UV purity=100%.

Example 11—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-3-(carbamoylmethyl)-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-iumbromide

2-(Aminomethyl)-3-(carbamoylmethyl)-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-iumhydrochloride bromide, Intermediate 43 (78%, 265 mg, 0.544 mmol) wasadded to a solution of lithium(1⁺) ion3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate 3 (60 mg,0.33 mmol), DIPEA (230 μl, 1.3 mmol) and HBTU (160 mg, 0.42 mmol) in DMF(2 ml). The resulting mixture was stirred at RT for 16 h thenconcentrated in vacuo to a thick red oil. The crude material thusobtained was purified by flash column chromatography on C18 (30 g). Thecolumn was eluted with MeCN:water+0.1% formic acid using the followinggradient (% MeCN, column volumes): 10%, 2 CV; 10-34%, 13 CV; 34-100%, 6CV; 100%, 3 CV. The desired fractions were combined and concentrated invacuo to afford the product as a pale brown solid (36 mg, 21%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.50 (s, 1H), 9.56 (t, J=5.5 Hz, 1H), 7.89(s, 1H), 7.80 (d, J=9.2 Hz, 1H), 7.64-7.57 (m, 2H), 7.50 (dd, J=3.7, 2.4Hz, 1H), 7.29 (dd, J=9.1, 2.3 Hz, 1H), 7.21 (s, 2H), 6.39 (dd, J=3.7,1.5 Hz, 1H), 5.40 (s, 2H), 4.99 (d, J=5.5 Hz, 2H), 4.71 (q, J=7.1 Hz,2H), 3.91 (s, 3H), 1.40 (t, J=7.2 Hz, 3H).

LC/MS (System C): m/z (ESI⁺)=423 [M⁺], Rt=1.39 min, UV purity=94%.

Example 12—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-6-methoxy-3-[2-(methylsulfanyl)ethyl]-1H-1,3-benzodiazol-3-iumiodide

A solution of2-(aminomethyl)-1-ethyl-6-methoxy-3-[2-(methylsulfanyl)ethyl]-1H-1,3-benzodiazol-3-iumiodide, Intermediate 45 (80%, 370 mg, 0.73 mmol) in DMF (1 ml) was addedto a solution of lithium(1⁺) ion3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate 3 (100 mg,0.54 mmol), DIPEA (380 μl, 2.2 mmol) and HBTU (270 mg, 0.71 mmol) in DMF(1 ml). The resulting mixture was stirred at RT for 21 h thenconcentrated in vacuo to a viscous red oil. The crude material wasdissolved in 2:1 DMSO:MeCN to a final volume of 1.5 ml then purified bypreparative HPLC (Method A). The desired fractions were combined andconcentrated in vacuo to a beige solid (47 mg). The material thusobtained was suspended in CH₂Cl₂ (2 ml). The supernatant was decantedoff from the solid via a pipette. The trituration procedure was repeatedthree more times then the solid was dried under vacuum to afford theproduct as a brown solid (23 mg, 7%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.52 (s, 1H), 9.68 (t, J=5.4 Hz, 1H), 7.96(d, J=9.2 Hz, 1H), 7.57 (d, J=2.3 Hz, 1H), 7.51 (dd, J=3.7, 2.5 Hz, 1H),7.38-7.11 (m, 3H), 6.40 (dd, J=3.8, 1.7 Hz, 1H), 5.07 (d, J=5.4 Hz, 2H),4.84 (t, J=7.2 Hz, 2H), 4.67 (q, J=7.1 Hz, 2H), 3.91 (s, 3H), 2.97 (t,J=7.2 Hz, 2H), 2.13 (s, 3H), 1.39 (t, J=7.2 Hz, 3H).

LC/MS (System C): m/z (ESI⁺)=440 [M⁺], Rt=1.96 min, UV purity=96%.

Example 13—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-3-(2-hydroxyethyl)-6-methoxy-1H-1,3-benzodiazol-3-iumformate

A mixture of2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 4 (92%, 70 mg, 0.28 mmol) and2-(aminomethyl)-1-ethyl-3-(2-hydroxyethyl)-6-methoxy-1H-1,3-benzodiazol-3-iumhydrochloride bromide, Intermediate 47 (125 mg, 0.341 mmol) in DMF (2ml) was stirred at RT for 16 h. The reaction mixture was concentrated invacuo then the resulting solid was suspended in MeCN (5 ml) withsonication. The solid was collected by filtration then washed with MeCN(15 ml) and dried under vacuum to afford a yellow solid (85 mg). Thesolid thus obtained was dissolved in DMSO then purified by preparativeHPLC (Method A, 4 separate injections). The desired fractions werecombined and concentrated in vacuo to afford the product as a yellowsolid (43 mg, 33%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.70 (s, 1H), 9.63 (t, J=5.4 Hz, 1H), 8.52(s, 1H), 7.97 (d, J=9.2 Hz, 1H), 7.55 (d, J=2.3 Hz, 1H), 7.50 (d, J=3.8Hz, 1H), 7.34-7.04 (m, 3H), 6.39 (d, J=3.8 Hz, 1H), 5.07 (d, J=5.4 Hz,2H), 4.79 (t, J=4.9 Hz, 2H), 4.65 (q, J=7.1 Hz, 2H), 3.91 (s, 3H), 3.80(t, J=4.9 Hz, 2H), 1.37 (t, J=7.2 Hz, 3H).

LC/MS (System C): m/z (ESI⁺)=410 [M⁺], Rt=1.51 min, UV purity=99%.

Example 14—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-3-{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}-6-methoxy-1H-1,3-benzodiazol-3-iumformate

A mixture of2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 4 (92%, 200 mg, 0.806 mmol) and2-(aminomethyl)-1-ethyl-3-{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}-6-methoxy-1H-1,3-benzodiazol-3-iumiodide, Intermediate 49 (84%, 554 mg, 1.00 mmol) in DMF (5 ml) wasstirred at RT for 1.5 h. The reaction mixture was concentrated in vacuothen the crude material was purified by flash column chromatography onC18 (60 g). The column was eluted with MeCN:water+0.1% formic acid usingthe following gradient (% MeCN, column volumes): 10%, 2 CV; 10-36%, 17CV; 36-100%, 1 CV; 100%, 3 CV. The desired fractions were combined andconcentrated in vacuo to give a brown/yellow oil. The material thusobtained was further purified by preparative HPLC using the followingmethod: Solvent A: Water+0.1% formic acid; Solvent B: MeCN+0.1% formicacid; Column: Waters Sunfire 30 mm×100 mm, 5 μm; Flowrate=40 ml/min;Gradient (time, Solvent B): 0 min, 2%; 2.5 min, 2%; 20.5 min, 15%; 21min, 100%; 23 min, 100%; 23.5 min, 5%. The desired fractions werecombined and concentrated in vacuo to afford the product as ayellow/orange solid (79 mg, 18%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.60 (s, 1H), 9.61 (t, J=5.4 Hz, 1H), 8.53(s, 1H), 7.96 (d, J=9.2 Hz, 1H), 7.55 (d, J=2.3 Hz, 1H), 7.51 (d, J=3.8Hz, 1H), 7.32-7.13 (m, 3H), 6.40 (d, J=3.7 Hz, 1H), 5.06 (d, J=5.5 Hz,2H), 4.90 (t, J=5.0 Hz, 2H), 4.66 (q, J=7.0 Hz, 2H), 3.91 (s, 3H), 3.82(d, J=4.9 Hz, 2H), 3.53-3.46 (m, 2H), 3.41-3.38 (m, 4H), 3.38-3.20 (m,2H+HDO), 1.36 (t, J=7.2 Hz, 3H).

LC/MS (System C): m/z (ESI⁺)=498 [M⁺], Rt=1.63 min, UV purity=98%.

Example 15—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-benzyl-3-methyl-1H-1,3-benzodiazol-3-iumformate

Iodomethane (22 μl, 0.35 mmol) was added to a solution of3-amino-N-[(1-benzyl-1H-1,3-benzodiazol-2-yl)methyl]-5H-pyrrolo[2,3-b]pyrazine-2-carboxamide,Intermediate 85 (35 mg, 0.088 mmol) in DMSO (1 ml). The resultingmixture was stirred at RT for 5 h. Additional iodomethane (40 μl, 0.64mmol) was added and the reaction mixture was left to stir for a further16 h at RT. The reaction mixture was diluted with MeCN to a final volumeof 1.5 ml then purified by preparative HPLC (Method A). The desiredfractions were combined and concentrated in vacuo to afford the productas a yellow solid (9.1 mg, 22%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.55 (s, 1H), 9.59 (t, J=5.4 Hz, 1H), 8.43(s, 1H), 8.09 (d, J=8.4 Hz, 1H), 7.76 (d, J=8.3 Hz, 1H), 7.69 (t, J=7.8Hz, 1H), 7.61 (t, J=7.8 Hz, 1H), 7.49 (dd, J=3.6, 2.4 Hz, 1H), 7.27-7.11(m, 7H), 6.36 (dd, J=3.7, 1.3 Hz, 1H), 5.96 (s, 2H), 5.14 (d, J=5.4 Hz,2H), 4.23 (s, 3H).

LC/MS (System C): m/z (ESI⁺)=412 [M⁺], Rt=1.82 min, UV purity=98%.

Example 16—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-3-benzyl-6-chloro-1-ethyl-1H-1,3-benzodiazol-3-iumbromide

A solution of2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 4 (60 mg, 0.26 mmol) and2-(aminomethyl)-3-benzyl-6-chloro-1-ethyl-1H-1,3-benzodiazol-3-iumbromide, Intermediate 51 (85%, 110 mg, 0.25 mmol) in DMF (2 ml) wasstirred at RT for 72 h. The resultant suspension was filtered then thesolid collected was dried under vacuum. The solid was re-suspended inMeCN (5 ml) then collected by filtration and dried under vacuum toafford a solid (70 mg). The solid was re-suspended in MeOH/MeCN/water(1:1:1, 3 ml) then filtered. The solid thus obtained was dried undervacuum to afford an orange solid (40 mg). The resultant solid wassuspended in DMSO:MeCN (2:1, 0.5 ml) then MeOH (0.5 ml) was added. Thesolid was collected by filtration and dried under vacuum to afford anorange solid (11 mg). The solid this obtained was dissolved MeCN:water(1:1, 1 ml) then lyophilised to afford the product as a yellow solid (9mg, 7%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.47 (s, 1H), 9.57 (t, J=5.3 Hz, 1H), 8.38(d, J=1.8 Hz, 1H), 7.78 (d, J=8.9 Hz, 1H), 7.68 (dd, J=8.9, 1.9 Hz, 1H),7.48 (dd, J=3.7, 2.5 Hz, 1H), 7.26-7.08 (m, 7H), 6.35 (dd, J=3.8, 1.7Hz, 1H), 5.95 (s, 2H), 5.15 (d, J=5.3 Hz, 2H), 4.74 (q, J=7.2 Hz, 2H),1.44 (t, J=7.2 Hz, 3H).

LC/MS (System C): m/z (ESI⁺)=460 [M(³⁵Cl)⁺], 462 [M(³⁷Cl)⁺], Rt=2.23min, UV purity=98%.

Example 17—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-3-benzyl-1-ethyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-iumchloride

A solution of2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 4 (50 mg, 0.22 mmol) and2-(aminomethyl)-3-benzyl-1-ethyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-iumhydrochloride bromide, Intermediate 53 (110 mg, 0.24 mmol) in DMF (1 ml)was stirred at RT for 16 h. The reaction mixture was concentrated invacuo then dissolved in MeCN:water (1:1, 2 ml). A precipitate formedwhich was collected by filtration, washed with MeCN:water (1:1) thendried under vacuum to afford the product as an orange solid (45 mg,39%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.48 (s, 1H), 9.58 (t, J=5.3 Hz, 1H), 8.69(s, 1H), 7.98 (s, 2H), 7.48 (dd, J=3.7, 2.5 Hz, 1H), 7.28-7.02 (m, 7H),6.35 (dd, J=3.8, 1.7 Hz, 1H), 6.01 (s, 2H), 5.20 (d, J=5.3 Hz, 2H), 4.86(d, J=7.3 Hz, 2H), 1.47 (t, J=7.2 Hz, 3H).

LC/MS (System C): m/z (ESI⁺)=494 [M⁺], Rt=2.34 min, UV purity=100%.

Example 18—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-3-benzyl-1-[2-oxo-2-(piperidin-1-yl)ethyl]-1H-1,3-benzodiazol-3-iumchloride

A solution of2-(aminomethyl)-1-benzyl-3-[2-oxo-2-(piperidin-1-yl)ethyl]-1H-1,3-benzodiazol-3-iumhydrochloride bromide, Intermediate 57 (76%, 191 mg, 0.303 mmol) in DMF(1 ml) was added to a solution of lithium(1⁺) ion3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate, Intermediate 3 (60 mg,0.33 mmol), DIPEA (230 μl, 1.3 mmol) and HBTU (160 mg, 0.42 mmol) in DMF(2 ml). The resulting mixture was stirred at RT for 16 h thenconcentrated in vacuo to a viscous red oil. The crude material wasdissolved in 2:1 DMSO:MeCN to a final volume of 1.5 ml then purified bypreparative HPLC (Method A). The desired fractions were combined andconcentrated in vacuo to afford the product as a pale beige solid (48mg, 26%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.46 (s, 1H), 9.45 (t, J=5.6 Hz, 1H), 7.98(d, J=8.3 Hz, 1H), 7.86 (d, J=7.9 Hz, 1H), 7.70-7.58 (m, 2H), 7.49 (dd,J=3.7, 2.5 Hz, 1H), 7.39-7.00 (m, 7H), 6.36 (dd, J=3.7, 1.7 Hz, 1H),6.07 (s, 2H), 5.79 (s, 2H), 5.11 (d, J=5.6 Hz, 2H), 3.55-3.49 (m, 2H),3.17-3.12 (m, 2H), 1.75-1.64 (m, 2H), 1.61-1.48 (m, 2H), 1.25-1.17 (m,2H).

LC/MS (System C): m/z (ESI⁺)=523 [M⁺], Rt=2.21 min, UV purity=99%.

Example 19—Synthesis of2-[({3-amino-6-methyl-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-6-methoxy-3-methyl-1H-1,3-benzodiazol-3-iumformate

CDI (61 mg, 0.37 mmol) was added to a solution of3-amino-6-methyl-5H-pyrrolo[2,3-b]pyrazine-2-carboxylic acid,Intermediate 9 (90%, 60 mg, 0.28 mmol) in DMF (2 ml). The reactionmixture was stirred at RT for 1 h.2-(aminomethyl)-1-ethyl-6-methoxy-3-methyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 25 (108 mg, 0.312 mmol) was added then the reactionwas left to stir for a further 16 h. The reaction mixture wasconcentrated in vacuo then purified by flash column chromatography onC18 (12 g). The column was eluted with MeCN:water+0.1% formic acid usingthe following gradient (% MeCN, column volumes): 0%, 3 CV; 0-20%, 14 CV;20-60% 3 CV; 60-100%, 1 CV, 100% 2 CV. The desired fractions werecombined and concentrated in vacuo to afford the product as a yellowsolid (35 mg, 28%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.42 (s, 1H), 9.57 (t, J=5.4 Hz, 1H), 8.51(s, 1H), 7.92 (d, J=9.1 Hz, 1H), 7.55 (d, J=2.3 Hz, 1H), 7.28 (dd,J=9.1, 2.3 Hz, 1H), 7.10 (s, 2H), 6.11 (s, 1H), 4.98 (d, J=5.4 Hz, 2H),4.64 (q, J=7.2 Hz, 2H), 4.11 (s, 3H), 3.90 (s, 3H), 2.37-2.34 (m, 3H),1.36 (t, J=7.2 Hz, 3H).

LC/MS (System C): m/z (ESI⁺)=394 [M⁺], Rt=1.80 min, UV purity=100%.

Example 20—Synthesis of2-[({3-amino-7-methyl-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-chloro-1,3-diethyl-1H-1,3-benzodiazol-3-iumformate

A mixture of2-(1H-imidazole-1-carbonyl)-7-methyl-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 13 (95%, 83 mg, 0.33 mmol) and2-(aminomethyl)-6-chloro-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide,Intermediate 34 (97%, 140 mg, 0.37 mmol) in DMF (3 ml) was stirred at RTfor 64 h. The reaction mixture was concentrated in vacuo then the crudematerial was purified by flash column chromatography on C18 (30 g). Thecolumn was eluted with MeCN:water+0.1% formic acid using the followinggradient (% MeCN, column volumes): 10%, 2 CV; 10-34%, 16 CV; 34-65%, 4CV; 65-100%, 2 CV; 100%, 1 CV. The desired fractions were combined andconcentrated in vacuo to remove most of the MeCN then lyophilised toafford the product as a yellow solid (44 mg, 25%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.21-11.16 (m, 1H), 9.49 (t, J=5.5 Hz, 1H),8.49 (s, 1H), 8.34 (d, J=1.8 Hz, 1H), 8.12 (d, J=8.9 Hz, 1H), 7.75 (dd,J=8.9, 1.9 Hz, 1H), 7.26 (dd, J=2.2, 1.2 Hz, 1H), 7.13 (s, 2H), 5.11 (d,J=5.4 Hz, 2H), 4.75-4.67 (m, 4H), 2.24 (d, J=1.1 Hz, 3H), 1.43-1.38 (m,6H).

LC/MS (System C): m/z (ESI⁺)=412 [M(³⁵Cl)⁺], 414 [M(³⁷Cl)⁺], Rt=2.07min, UV purity=96%.

Example 21—Synthesis of2-[({3-amino-7-chloro-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-chloro-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide

A mixture of7-chloro-2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 16 (95%, 160 mg, 0.58 mmol) and2-(aminomethyl)-6-chloro-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide,Intermediate 34 (97%, 220 mg, 0.58 mmol) in DMF (4 ml) was stirred at RTfor 64 h. The reaction mixture was concentrated in vacuo then the crudematerial was purified by flash column chromatography on C18 (30 g). Thecolumn was eluted with MeCN:water+0.1% formic acid using the followinggradient (% MeCN, column volumes): 10%, 2 CV; 10-33%, 13 CV; 33-41%, 2CV; 41-70%, 5 CV; 70%, 3 CV. The desired fractions were combined andconcentrated in vacuo to remove most of the MeCN then lyophilised toafford the product as a yellow/orange solid (161 mg, 48%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.80 (s, 1H), 9.51 (t, J=5.3 Hz, 1H), 8.34(d, J=1.8 Hz, 1H), 8.12 (d, J=8.9 Hz, 1H), 7.75 (dd, J=8.9, 1.9 Hz, 1H),7.69 (s, 1H), 7.37 (s, 2H), 5.12 (d, J=5.4 Hz, 2H), 4.77-4.61 (m, 4H),1.44-1.37 (m, 6H).

LC/MS (System C): m/z (ESI⁺)=432 [M(³⁵Cl₂)⁺], 434 [M(³⁵Cl³⁷Cl)⁺], 436[M(³⁷Cl₂)⁺], Rt=2.14 min, UV purity=97%.

Example 22—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumformate

A mixture of2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 4 (98%, 369 mg, 1.59 mmol) and2-(aminomethyl)-6-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 59 (82%, 819 mg, 1.37 mmol) in DMF (8 ml) wasstirred at RT for 90 h. The reaction mixture was concentrated in vacuothen the crude material was purified by flash column chromatography onC18 (120 g). The column was eluted with MeCN:water+0.1% formic acidusing the following gradient (% MeCN, column volumes): 10%, 2 CV;10-32%, 11 CV; 32-100%, 3 CV; 100% 2 CV. The desired fractions werecombined and concentrated in vacuo to remove most of the MeCN thenlyophilised to afford the product as a yellow solid (382 mg, 47%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.57 (s, 1H), 9.70 (t, J=5.2 Hz, 1H), 8.44(s, 1H), 7.95 (d, J=9.1 Hz, 1H), 7.63 (s, 1H), 7.54-7.49 (m, 1H),7.31-7.13 (m, 3H), 7.08 (t, J=5.4 Hz, 1H), 6.42 (d, J=3.5 Hz, 1H), 5.04(d, J=5.3 Hz, 2H), 4.66 (q, J=7.0 Hz, 4H), 4.12 (t, J=5.8 Hz, 2H), 3.36(m, 2H+HDO), 1.41-1.37 (m, 15H).

LC/MS (System C): m/z (ESI⁺)=523 [M⁺], Rt=2.26 min, UV purity=95%.

Example 23—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumformate

A mixture of2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 4 (98%, 295 mg, 1.27 mmol) and2-(aminomethyl)-6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 64 (91%, 710 mg, 1.28 mmol) in DMF (5 ml) wasstirred at RT for 16 h. Additional DMF (3 ml) was added then thereaction was left to stir for a further 72 h. The reaction mixture wasconcentrated in vacuo then the crude material was purified by flashcolumn chromatography on C18 (60 g). The column was eluted withMeCN:water+0.1% formic acid using the following gradient (% MeCN, columnvolumes): 10%, 2 CV; 10-34%, 16 CV; 34-100%, 3 CV; 100% 2 CV. Thedesired fractions were combined and concentrated in vacuo to afford theproduct as a viscous orange oil (345 mg, 46%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.53 (s, 1H), 9.69 (t, J=5.4 Hz, 1H), 8.46(s, 1H), 7.94 (d, J=9.1 Hz, 1H), 7.56 (d, J=2.0 Hz, 1H), 7.51 (dd,J=3.6, 1.9 Hz, 1H), 7.26 (dd, J=9.1, 2.2 Hz, 1H), 7.20 (s, 2H),6.93-6.88 (m, 1H), 6.41 (d, J=3.2 Hz, 1H), 5.03 (d, J=5.4 Hz, 2H),4.72-4.58 (m, 4H), 4.12 (t, J=6.1 Hz, 2H), 3.11 (q, J=6.6 Hz, 2H), 1.89(p, J=6.4 Hz, 2H), 1.41-1.35 (m, 15H).

LC/MS (System C): m/z (ESI⁺)=537 [M⁺], Rt=2.39 min, UV purity=100%.

Example 24—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-{[(tert-butoxy)carbonyl]amino}propyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumformate

2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 4 (252 mg, 1.11 mmol) was added to a solution of2-(aminomethyl)-6-(3-{[(tert-butoxy)carbonyl]amino}propyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 69 (80%, 540 mg, 0.884 mmol) in DMF (12 ml). Theresulting solution was stirred at RT for 16 h then for at 30° C. for 2h. The resultant solution was concentrated in vacuo. The crude materialwas purified by flash column chromatography on C18 (30 g). The columnwas eluted with MeCN:water+0.1% formic acid using the following gradient(% MeCN, column volumes): 10%, 2 CV; 10-34%, 16 CV; 34-100%, 3 CV, 100%2 CV. The desired fractions were combined and concentrated in vacuo toafford the product as a light brown solid (235 mg, 40%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.55 (s, 1H), 9.70 (t, J=5.3 Hz, 1H), 8.35(s, 1H), 7.96 (d, J=8.6 Hz, 1H), 7.89 (s, 1H), 7.55-7.50 (m, 2H), 7.19(s, 2H), 6.89 (t, J=5.3 Hz, 1H), 6.41 (d, J=3.6 Hz, 1H), 5.05 (d, J=5.2Hz, 2H), 4.66 (dd, J=7.1, 2.5 Hz, 4H), 2.94 (q, J=6.6 Hz, 2H), 2.78 (t,J=7.5 Hz, 2H), 1.79-1.71 (m, 2H), 1.40 (dt, J=7.1, 3.7 Hz, 6H), 1.38 (s,9H).

LC/MS (System C): m/z (ESI⁺)=521 [M⁺], Rt=2.34 min, UV purity=97%.

Example 25—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide

2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 4 (251 mg, 1.10 mmol) was added to a solution of2-(aminomethyl)-6-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 73 (72%, 605 mg, 0.847 mmol) in DMF (15 ml). Theresulting solution was stirred at RT for 24 h. The reaction mixture wasconcentrated in vacuo then the crude material was purified by flashcolumn chromatography on C18 (60 g). The column was eluted withMeCN:water+0.1% formic acid using the following gradient (% MeCN, columnvolumes): 0%, 2 CV; 0-17%, 5 CV; 17-39%, 6 CV; 39%, 2 CV; 39-100%, 4 CV;100%, 1 CV. The desired fractions were combined and lyophilised toafford the product as a yellow solid (315 mg, 52%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.56 (s, 1H), 9.70 (t, J=5.2 Hz, 1H), 7.97(d, J=8.6 Hz, 1H), 7.96 (s, 1H), 7.65-7.57 (m, 1H), 7.51 (dd, J=3.7, 1.7Hz, 1H), 7.19 (s, 2H), 6.41 (d, J=3.5 Hz, 1H), 5.05 (d, J=5.3 Hz, 2H),4.73-4.62 (m, 4H), 4.19-4.09 (m, 2H), 3.00-2.90 (m, 1H), 2.85 (br. s,2H), 1.81 (d, J=12.2 Hz, 2H), 1.65 (qd, J=12.8, 4.3 Hz, 2H), 1.47-1.36(m, 15H).

LC/MS (System C): m/z (ESI⁺)=547 [M⁺], Rt=2.66 min, UV purity=95%.

Example 26—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(1-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1H-pyrazol-4-yl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumformate

2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 4 (312 mg, 1.33 mmol) was added to a solution of2-(aminomethyl)-6-(1-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1H-pyrazol-4-yl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 76 (83%, 880 mg, 1.26 mmol) in DMF (10 ml). Theresulting solution was stirred at RT for 64 h. The reaction mixture wasconcentrated in vacuo then the crude material was purified by flashcolumn chromatography on C18 (60 g). The column was eluted withMeCN:water+0.1% formic acid using the following gradient (% MeCN, columnvolumes): 0%, 2 CV; 0-11%, 5 CV; 11-40%, 9 CV; 40%, 2 CV. The desiredfractions were combined and concentrated in vacuo to afford the productas a yellow solid (525 mg, 60%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.58 (s, 1H), 9.72 (t, J=5.3 Hz, 1H), 8.50(s, 1H), 8.33 (s, 1H), 8.24 (s, 1H), 8.11 (s, 1H), 8.04 (d, J=8.7 Hz,1H), 7.95-7.89 (m, 1H), 7.52 (dd, J=3.6, 2.3 Hz, 1H), 7.23 (s, 2H), 6.41(d, J=2.7 Hz, 1H), 5.06 (d, J=5.2 Hz, 2H), 4.68 (q, J=7.0 Hz, 4H), 4.39(ddt, J=11.4, 7.8, 3.9 Hz, 1H), 4.06 (br. s, 2H), 2.94 (br. s, 2H),2.11-2.03 (m, 2H), 1.82-1.78 (m, 2H), 1.47-1.38 (m, 15H).

LC/MS (System C): m/z (ESI⁺)=613 [M⁺], Rt=2.59 min, UV purity=94%.

Example 27—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(2-aminoethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrochloride chloride

HCl solution in dioxane (4.0 M, 140 μl, 0.56 mmol) was added to asolution of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumformate, Example 22 (95%, 39 mg, 0.065 mmol) in MeCN (1 ml). Theresulting mixture was stirred at RT for 50 min then concentrated under astream of nitrogen. The resulting residue was suspended in MeCN (1 ml).The solid was collected by filtration then washed with MeCN and driedunder vacuum to afford the product as an orange solid (20 mg, 64%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.54 (s, 1H), 9.71 (d, J=5.6 Hz, 1H), 8.13(s, 3H), 8.00 (d, J=9.2 Hz, 1H), 7.66 (d, J=2.1 Hz, 1H), 7.53 (dd,J=3.7, 2.5 Hz, 1H), 7.32 (dd, J=9.1, 2.3 Hz, 1H), 7.21 (s, 2H), 6.42(dd, J=3.8, 1.7 Hz, 1H), 5.05 (d, J=5.4 Hz, 2H), 4. 72-4.56 (m, 5H),4.40-4.31 (m, 3H), 1.42-1.38 (m, 6H).

LC/MS (System C): m/z (ESI⁺)=423 [M⁺], Rt=0.94 min, ELS purity=100%.

Example 28—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-aminopropoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrochloride chloride

HCl solution in dioxane (4.0 M, 1.6 ml, 6.4 mmol) was added to asolution of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumformate, Example 23 (382 mg, 0.656 mmol) in MeCN (10 ml). The resultingmixture was stirred at RT for 45 min then concentrated in vacuo toafford the product as an orange solid (292 mg, 84%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.55 (s, 1H), 9.70 (t, J=5.3 Hz, 1H), 8.07(s, 3H), 7.97 (d, J=9.1 Hz, 1H), 7.62 (d, J=2.1 Hz, 1H), 7.51 (dd,J=3.7, 2.5 Hz, 1H), 7.29 (m, 3H), 6.41 (dd, J=3.8, 1.7 Hz, 1H), 5.04 (d,J=5.3 Hz, 2H), 4.70-4.63 (m, 4H), 4.24 (t, J=6.2 Hz, 2H), 3.03-2.92 (m,2H), 2.09 (p, J=6.4 Hz, 2H), 1.39 (t, J=7.2 Hz, 6H).

LC/MS (System C): m/z (ESI⁺)=437 [M⁺], Rt=1.02 min, UV purity=96%.

Example 29—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-aminopropyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrochloride chloride

HCl solution in dioxane (4.0 M, 31 μl, 0.12 mmol) was added to asolution of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-{[(tert-butoxy)carbonyl]amino}propyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide, Example 24 (40 mg, 0.06 mmol) in MeCN (1 ml). The resultingmixture was stirred at RT for 30 min then more HCl solution in dioxane(4.0 M, 15 μl, 0.060 mmol) was added. The reaction mixture was stirredat RT for a further 20 min then concentrated under a stream of nitrogen.The resulting residue was dried under vacuum to afford the product as ayellow solid (27 mg, 84%).

¹H NMR (500 MHz, CD₃OD) δ 7.92 (d, J=8.6 Hz, 1H), 7.88 (s, 1H), 7.61(dd, J=8.6, 1.3 Hz, 1H), 7.40 (d, J=3.8 Hz, 1H), 6.45 (d, J=3.8 Hz, 1H),5.12 (s, 2H), 4.82-4.75 (m, 4H), 3.04-2.96 (m, 4H), 2.08 (p, J=7.8 Hz,2H), 1.60-1.52 (m, 6H).

LC/MS (System C): m/z (ESI⁺)=421 [M⁺], Rt=1.03 min, UV purity=95%.

Example 30—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-(piperidin-4-yl)-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

HCl solution in dioxane (4.0 M, 0.27 ml, 1.1 mmol) was added to asolution of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide, Example 25 (95%, 300 mg, 0.42 mmol) in MeCN (15 ml). Theresulting solution was stirred at RT for 16 h then HCl solution indioxane (4.0 M, 0.27 ml, 1.1 mmol) was added. The reaction was stirredat RT for a further 4 h then concentrated in vacuo then the residue wasazeotroped with MeCN (2×30 ml). The resulting solid was suspended inMeCN (30 ml) then filtered, rinsed with MeCN and dried under vacuum toafford the product as a brown solid (90 mg). The remaining material onthe filter paper was dissolved in MeOH and combined with the filtratethen concentrated in vacuo to afford a brown viscous oil (125 mg). Theoil thus obtained was further purified by preparative HPLC (Method B).The desired fractions were combined and lyophilised to afford a brownsolid (35 mg). The material thus obtained was combined with the solidobtained by filtration (90 mg) then the combined material was dissolvedin MeCN:water (1:9, 3 ml) and lyophilised to afford a brown solid (125mg). The material was re-dissolved in MeCN:water (1:9, 3 ml) andlyophilised once more to afford a brown solid (110 mg). The materialthus obtained was further purified by flash column chromatography on C18(12 g). The column was eluted with MeCN:water+0.1% TFA using thefollowing gradient (% MeCN, column volumes): 0%, 2 CV; 0-5%, 2 CV;5-15%, 3 CV; 15-25%, 3 CV; 25-51%, 3 CV; 51-100%, 2 CV; 100%, 1 CV. Thedesired fractions were combined and lyophilised to afford a yellow solid(70 mg). The material thus obtained was further purified by HPLC (MethodC). The desired fractions were combined and lyophilised to afford anorange solid (63 mg). The material thus obtained was further purified byflash column chromatography on C18 (30 g). The column was eluted withMeCN:water+0.1% TFA using the following gradient (% MeCN, columnvolumes): 5%, 3 CV; 5-14%, 7 CV; 14%, 4 CV; 14-20%, 5 CV; 20-26%, 1 CV;26-100%, 2 CV; 100%, 2 CV. The desired fractions were combined andlyophilised to afford the product as a yellow solid (27 mg, 9%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.54 (s, 1H), 9.72 (t, J=5.3 Hz, 1H), 8.73(d, J=9.5 Hz, 1H), 8.46 (d, J=10.4 Hz, 1H), 8.04 (d, J=8.7 Hz, 1H), 7.88(s, 1H), 7.57 (dd, J=8.7, 1.2 Hz, 1H), 7.53 (dd, J=3.7, 2.5 Hz, 1H),7.22 (s, 2H), 6.42 (dd, J=3.8, 1.7 Hz, 1H), 5.07 (d, J=5.3 Hz, 2H),4.75-4.65 (m, 4H), 3.49-3.40 (m, 2H+HDO), 3.16-2.99 (m, 3H), 2.10-2.01(m, 2H), 1.90 (qd, J=13.4, 3.8 Hz, 2H), 1.40 (t, 6H).

LC/MS (System C): m/z (ESI⁺)=447 [M⁺], Rt=0.98 min, UV purity=100%.

Example 31—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]-1H-1,3-benzodiazol-3-iumhydrochloride iodide

HCl solution in dioxane (4.0 M, 420 μl, 1.7 mmol) was added to asolution of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(1-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1H-pyrazol-4-yl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumformate, Example 26 (94%, 500 mg, 0.71 mmol) in MeCN. The reactionmixture was stirred at RT for 40 h then more HCl solution in dioxane(4.0 M, 200 μl, 0.80 mmol) was added. The reaction mixture was stirredat RT for a further 2 h then concentrated in vacuo. The residue wasazeotroped from MeCN then dried under vacuum. The residue thus obtainedwas re-suspended in MeCN (20 ml) then HCl solution in dioxane (4.0 M,420 μl, 1.7 mmol) was added. The reaction was stirred at RT for 88 h.The resulting suspension was filtered then the solid was dried undervacuum, re-dissolved in 1:1 MeCN:water (4 ml) then lyophilised to affordthe product as a brown solid (450 mg, 90%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.57 (s, 1H), 9.71 (t, J=5.3 Hz, 1H),9.40-9.31 (m, 1H), 9.30-9.20 (m, 1H), 8.49 (s, 1H), 8.30 (s, 1H), 8.16(s, 1H), 8.04 (d, J=8.7 Hz, 1H), 7.97-7.92 (m, 1H), 7.51 (dd, J=3.7, 2.5Hz, 1H), 6.41 (dd, J=3.7, 1.7 Hz, 1H), 5.07 (d, J=5.2 Hz, 2H), 4.73-4.66(m, 4H), 4.57-4.50 (m, 1H), 3.38 (d, J=12.3 Hz, 2H), 3.10 (dd, J=10.7Hz, 2H), 2.31-2.18 (m, 4H), 1.43 (dt, J=12.1, 7.2 Hz, 6H).

LC/MS (System C): m/z (ESI⁺)=513 [M⁺], Rt=1.20 min, UV purity=97%.

Example 32—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(2-carbamimidamidoethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumformate

DIPEA (18 μl, 0.10 mmol) was added to a mixture of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(2-aminoethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrochloride chloride, Example 27 (18 mg, 0.036 mmol) and1H-1,2,4-triazole-1-carboximidamide hydrochloride (1:1) (5.5 mg, 0.037mmol) in DMF (1 ml). The resulting mixture was stirred at RT for 3.5 h.Additional 1H-1,2,4-triazole-1-carboximidamide hydrochloride (1:1) (5.5mg, 0.037 mmol) was added and the reaction allowed to continue for afurther 18 h. The reaction mixture was concentrated in vacuo then thecrude material was purified by flash column chromatography on C18 (12g). The column was eluted with MeCN:water+0.1% formic acid using thefollowing gradient (% MeCN, column volumes): 0%, 2 CV; 0-1%, 1 CV;1-70%, 6 CV; 70%, 1 CV; 70-100% 3 CV; 100% 2 CV. The desired fractionswere combined and concentrated in vacuo to afford an orange solid (17mg). The material thus obtained was further purified by preparative HPLC(Method A). The desired fractions were combined and concentrated invacuo to afford the product as a yellow solid (1.7 mg, 9%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.55 (s, 1H), 9.70 (t, J=5.3 Hz, 1H), 8.59(s, 1H), 8.44 (s, 1H), 7.97 (d, J=9.1 Hz, 1H), 7.75-7.62 (m, 4H), 7.51(d, J=3.8 Hz, 1H), 7.35-7.09 (m, 3H), 6.41 (d, J=3.8 Hz, 1H), 5.04 (d,J=5.1 Hz, 2H), 4.70-4.62 (m, 4H), 4.21 (t, J=5.1 Hz, 2H), 3.60-3.54 (m,2H), 1.43-1.35 (m, 6H).

LC/MS (System C): m/z (ESI⁺)=465 [M⁺], Rt=1.07 min, UV purity=100%.

Example 33—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-carbamimidamidopropyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumchloride

1H-1,2,4-triazole-1-carboximidamide hydrochloride (1:1) (13 mg, 0.090mmol) was added to a suspension of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-aminopropyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrochloride chloride, Example 29 (50 mg, 0.10 mmol) in CH₂Cl₂ (4 ml).DIPEA (22 μl, 0.13 mmol) was added then the resulting suspension wasstirred at RT for 1 h. The reaction was concentrated under a stream ofnitrogen then DMF (4 ml) was added. The resulting solution was stirredat RT for 3 h. A further portion of 1H-1,2,4-triazole-1-carboximidamidehydrochloride (1:1) (13 mg, 0.090 mmol) and by DIPEA (22 μl, 0.13 mmol)was added then the reaction mixture was left to stir for a further 16 h.The reaction mixture was concentrated in vacuo then the crude materialwas purified by flash column chromatography on C18 (12 g). The columnwas eluted with MeCN:water+0.1% formic acid using the following gradient(% MeCN, column volumes): 0%, 5 CV; 0-42%, 17 CV; 42-65%, 3 CV; 65-100%1 CV; 100% 1 CV. The desired fractions were combined and concentrated invacuo. The material thus obtained was further purified by preparativeHPLC (Method A). The desired fractions were combined and concentrated invacuo to afford the product as a yellow solid (10 mg, 19%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.56 (s, 1H), 9.73 (t, J=5.3 Hz, 1H), 8.82(s, 1H), 8.42 (s, 1H), 7.98 (d, J=8.5 Hz, 1H), 7.94 (s, 1H), 7.60-7.54(m, 3H), 7.52 (dd, J=3.7, 2.4 Hz, 1H), 7.21 (s, 2H), 6.44-6.39 (m, 1H),5.05 (d, J=5.2 Hz, 2H), 4.68 (q, J=6.9 Hz, 4H), 3.10 (d, J=5.8 Hz, 2H),2.87-2.81 (m, 2H), 1.89-1.81 (m, 2H), 1.40 (td, J=7.2, 4.0 Hz, 6H).

LC/MS (System C): m/z (ESI⁺)=463 [M⁺], Rt=1.16 min, UV purity=98%.

Example 34—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide

A suspension of2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 4 (1.26 g, 5.52 mmol) and2-(aminomethyl)-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrobromide bromide, Intermediate 83 (2.26 g, 5.52 mmol) in DMF (11 ml)was stirred at RT for 16 h. The reaction mixture was filtered then theresulting solid was washed with MeCN and dried under vacuum to afford ayellow solid (0.89 g). The filtrate was filtered then the resultingsolid was dried under vacuum to afford a yellow solid (0.42 g). Thefiltrate was concentrated in vacuo then the residue was suspended inMeCN/water (4:1, 5 ml) then filtered and the resulting solid was driedunder vacuum to afford a yellow solid (1.15 g). The solids thus obtainedwere combined as an MeCN suspension then concentrated in vacuo and driedunder vacuum to afford the product as a yellow solid (2.46 g, 91%).

¹H NMR (500 MHz, DMSO-d₆) δ 13.50 (s, 1H), 11.52 (s, 1H), 9.72 (t, J=5.3Hz, 1H), 8.60 (s, 1H), 8.22 (dd, J=8.7, 1.3 Hz, 1H), 8.17 (d, J=8.7 Hz,1H), 7.52 (dd, J=3.8, 2.5 Hz, 1H), 7.20 (s, 2H), 6.42 (dd, J=3.8, 1.7Hz, 1H), 5.10 (d, J=5.3 Hz, 2H), 4.80 (q, J=7.2 Hz, 2H), 4.73 (q, J=7.2Hz, 2H), 1.42 (t, J=7.2 Hz, 6H).

LC/MS (System C): m/z (ESI⁺)=408 [M⁺], Rt=1.39 min, UV purity=100%.

Example 35—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3-{[(tert-butoxy)carbonyl]amino}propyl)carbamoyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumformic acid formate

tert-Butyl N-(3-aminopropyl)carbamate (89 mg, 0.51 mmol) was added to asolution of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (250 mg, 0.51 mmol), HBTU (210 mg, 0.56 mmol), and4-methylmorpholine (0.13 ml, 1.0 mmol) in DMF (2.5 ml). The reaction wasstirred at RT for 16 h. The reaction mixture was concentrated in vacuothen the crude material was purified by flash column chromatography onC18 (30 g). The column was eluted with MeCN:water+0.1% formic acid usingthe following gradient (% MeCN, column volumes): 5%, 2 CV; 5-21%, 3 CV;21%, 1 CV; 21-29%, 2 CV; 29%, 3 CV; 29-100%, 14 CV. The desiredfractions were combined and concentrated in vacuo to afford the productas a yellow solid (205 mg, 60%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.54 (s, 1H), 9.72 (t, J=5.3 Hz, 1H), 8.73(t, J=5.5 Hz, 1H), 8.47 (s, 1H), 8.23 (s, 2H), 8.19-8.09 (m, 2H),7.57-7.47 (m, 1H), 7.20 (s, 2H), 6.84 (t, J=5.6 Hz, 1H), 6.47-6.38 (m,1H), 5.09 (d, J=5.3 Hz, 2H), 4.81-4.61 (m, 4H), 3.37-3.21 (m, 2H+HDO),3.09-2.96 (m, 2H), 1.75-1.62 (m, 2H), 1.51-1.31 (m, 15H).

LC/MS (System D): m/z (ESI⁺)=564 [M⁺], Rt=2.35 min, UV purity=98%.

Example 36—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-({1-[(tert-butoxy)carbonyl]piperidin-4-yl}carbamoyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide

tert-Butyl 4-aminopiperidine-1-carboxylate (82 mg, 0.41 mmol) was addedto a solution of2-[[(3-amino-5H-pyrrolo[2,3-b]pyrazine-2-carbonyl)amino]methyl]-1,3-diethyl-benzimidazol-1-ium-5-carboxylicacid bromide, Example 34 (200 mg, 0.410 mmol), HBTU (171 mg, 0.450 mmol)and 4-methylmorpholine (104 μl, 0.820 mmol) in DMF (2 ml). The reactionwas stirred at RT for 64 h then concentrated in vacuo. The crudematerial was purified by flash column chromatography on C18 (30 g). Thecolumn was eluted with MeCN:water+0.1% formic acid using the followinggradient (% MeCN, column volumes): 5%, 3 CV; 5-20%, 3 CV; 20%, 4 CV;20-46%, 5 CV; 46%, 3 CV; 46-56%, 2 CV; 56-94%, 2 CV. The desiredfractions were combined and concentrated in vacuo to afford the productas an orange solid (240 mg, 87%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.53 (s, 1H), 9.72 (t, J=5.3 Hz, 1H), 8.51(d, J=7.8 Hz, 1H), 8.45 (s, 1H), 8.17-8.11 (m, 2H), 7.55-7.48 (m, 1H),7.32-7.07 (m, 2H), 6.45-6.39 (m, 1H), 5.10 (d, J=5.3 Hz, 2H), 4.80-4.65(m, 4H), 4.13-3.86 (m, 3H), 2.97-2.78 (m, 2H), 1.91-1.79 (m, 2H),1.52-1.36 (m, 17H).

LC/MS (System D): m/z (ESI⁺)=590 [M⁺], Rt=2.75 min, UV purity=100%.

Example 37—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{[(tert-butoxy)carbonyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide

A solution of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (200 mg, 0.410 mmol), HBTU (171 mg, 0.451 mmol), and4-methylmorpholine (104 μl, 0.819 mmol) in DMF (2 ml) was stirred at RTfor 5 min. tert-Butyl N-(piperidin-4-yl)carbamate (82 mg, 0.41 mmol) wasadded then the reaction mixture was stirred at RT for 16 h. The reactionmixture was concentrated in vacuo then the crude material was purifiedby flash column chromatography on C18 (30 g). The column was eluted withMeCN:water+0.1% TFA using the following gradient (% MeCN, columnvolumes): 5%, 2 CV; 5-25%, 4 CV; 25%, 1 CV; 25-48%, 5 CV; 48%, 1 CV;48-57%, 2 CV; 57-100%, 2 CV; 100%, 1 CV. The desired fractions werecombined and concentrated in vacuo to afford the product as a yellowsolid (135 mg, 48%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.53 (s, 1H), 9.78-9.65 (m, 1H), 8.52 (d,J=7.7 Hz, 1H), 8.45 (s, 1H), 8.15 (s, 2H), 7.57-7.46 (m, 1H), 7.20 (s,2H), 6.46-6.36 (m, 1H), 5.16-5.01 (m, 2H), 4.80-4.65 (m, 4H), 4.13-3.86(m, 3H), 2.89 (s, 2H), 1.90-1.78 (m, 2H), 1.52-1.34 (m, 17H).

LC/MS (System C): m/z (ESI⁺)=590 [M⁺], Rt=2.20 min, UV purity=98%.

Example 38—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{[2-(4-{[(tert-butoxy)carbonyl]amino}piperidin-1-yl)ethyl]carbamoyl}-1,3-diethyl-1H-1,3-benzodiazol-3-iumformate

A solution of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (220 mg, 0.45 mmol), and CDI (100 mg, 0.68 mmol) inDMF (5 ml) was stirred at RT for 0.5 h. Additional CDI (40 mg, 0.25mmol) was added and the reaction was stirred at RT for a further 0.5 h.A solution of tert-butyl N-[1-(2-aminoethyl)-4-piperidyl]carbamate (139mg, 0.57 mmol) in DMF (5 ml) was added then the resulting mixture wasstirred at RT for 10 min. The reaction mixture was concentrated in vacuothen the crude material was purified by flash column chromatography onC18 (30 g). The column was eluted with MeCN:water+0.1% formic acid usingthe following gradient (% MeCN, column volumes): 10%, 2 CV; 10-27%, 13CV; 27-34%, 3 CV; 34-59%, 4 CV; 59-100%, 1 CV; 100%, 2 CV. The desiredfractions were combined and concentrated in vacuo to afford the productas a yellow solid (261 mg, 85%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.54 (s, 1H), 9.72 (t, J=5.3 Hz, 1H), 8.84(t, J=5.4 Hz, 1H), 8.49 (s, 1H), 8.24 (s, 2H), 8.14 (s, 2H), 7.51 (dd,J=3.7, 2.2 Hz, 1H), 7.20 (s, 2H), 6.45-6.38 (m, 1H), 5.09 (d, J=5.3 Hz,2H), 4.79-4.65 (m, 4H), 3.82 (d, J=12.4 Hz, 2H), 3.48-3.41 (m, 2H+HDO),2.84-2.74 (m, 4H), 2.68-2.64 (m, 1H), 1.82-1.74 (m, 2H), 1.47-1.40 (m,6H), 1.37 (s, 9H), 1.18-1.08 (m, 2H).

LC/MS (System C): m/z (ESI⁺)=633 [M⁺], Rt=1.43 min, UV purity=99%.

Example 39—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3-aminopropyl)carbamoyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrochloride chloride

HCl solution in dioxane (4.0 M, 0.35 ml, 1.4 mmol) was added to asolution of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3-{[(tert-butoxy)carbonyl]amino}propyl)carbamoyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumformic acid formate, Example 35 (180 mg, 0.275 mmol) in MeCN (3 ml). Thereaction was stirred at RT for 1 h then concentrated in vacuo. Theresidue was azeotroped from MeCN (×2) to afford an orange solid. Thesolid thus obtained was dissolved in 1:1 MeCN:water then lyophilised toafford a yellow solid. The lyophilised solid was suspended in MeCN (1ml) with sonication. The resulting suspension was left to settle thenthe supernatant was decanted off with a pipette. The trituration processwas repeated once more then the solid was dried under vacuum to affordthe product as an orange solid (110 mg, 75%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.54 (s, 1H), 9.73 (t, J=5.3 Hz, 1H),9.18-9.08 (m, 1H), 8.63 (s, 1H), 8.23-8.12 (m, 2H), 8.05-7.81 (m, 3H),7.59-7.46 (m, 1H), 7.46-6.95 (m, 1H), 6.48-6.38 (m, 1H), 5.21-5.02 (m,2H), 4.87-4.62 (m, 4H), 3.47-3.27 (m, 2H+HDO), 2.98-2.81 (m, 2H),1.96-1.83 (m, 2H), 1.54-1.36 (m, 6H).

LC/MS (System D): m/z (ESI⁺)=464 [M⁺], Rt=1.17 min, UV purity=100%.

Example 40—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-[(piperidin-4-yl)carbamoyl]-1H-1,3-benzodiazol-3-iumhydrochloride chloride

HCl solution in dioxane (4.0 M, 0.37 ml, 1.5 mmol) was added to asolution of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-({1-[(tert-butoxy)carbonyl]piperidin-4-yl}carbamoyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 36 (210 mg, 0.313 mmol) in MeCN (3 ml). The reactionwas stirred at RT for 1 h. The reaction mixture was concentrated invacuo then azeotroped with MeCN (×2). The residue was re-dissolved inMeCN:water then lyophilised. The material thus obtained was suspended inMeCN (1 ml) with sonication. The supernatant was decanted off then theprocess was repeated with more MeCN (1 ml). The solid thus obtained wasdried under vacuum to afford the product as an orange solid (85 mg,48%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.55 (s, 1H), 9.73 (t, J=5.3 Hz, 1H),9.00-8.80 (m, 3H), 8.63 (s, 1H), 8.24-8.09 (m, 2H), 7.56-7.47 (m, 1H),7.47-6.91 (m, 1H), 6.46-6.37 (m, 1H), 5.16-5.04 (m, 2H), 4.82-4.66 (m,4H), 4.20-4.04 (m, 1H), 3.56-3.19 (m, 2H+HDO), 3.11-2.94 (m, 2H),2.07-1.96 (m, 2H), 1.94-1.80 (m, 2H), 1.52-1.36 (m, 6H).

LC/MS (System D): m/z (ESI⁺)=490 [M⁺], Rt=1.34 min, UV purity=100%.

Example 41—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-aminopiperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrochloride iodide

HCl solution in dioxane (4.0 M, 0.25 ml, 1.0 mmol) was added to asolution of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{[(tert-butoxy)carbonyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 37 (135 mg, 0.201 mmol) in MeCN (3 ml). The reactionmixture was stirred at RT for 20 min then concentrated in vacuo. Theresidue was azeotroped from MeCN (×2) then re-dissolved in water andlyophilised to afford the product as an orange solid (123 mg, 99%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.55 (s, 1H), 9.73 (t, J=5.3 Hz, 1H),8.99-8.76 (m, 3H), 8.62 (s, 1H), 8.25-8.10 (m, 2H), 7.52 (dd, J=3.8, 2.5Hz, 1H), 7.18 (s, 1H), 6.47-6.37 (m, 1H), 5.10 (d, J=5.3 Hz, 2H), 4.74(m, 4H), 4.12 (m, 1H), 3.40-3.25 (m, 2H+HDO), 3.14-2.94 (m, 2H),2.07-1.98 (m, 2H), 1.93-1.80 (m, 2H), 1.43 (m, 6H).

LC/MS (System C): m/z (ESI⁺)=490 [M⁺], Rt=0.98 min, UV purity=97%.

Example 42—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{[2-(4-aminopiperidin-1-yl)ethyl]carbamoyl}-1,3-diethyl-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

HCl solution in dioxane (4.0 M, 1.6 ml, 6.4 mmol) was added to asuspension of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{[2-(4-{[(tert-butoxy)carbonyl]amino}piperidin-1-yl)ethyl]carbamoyl}-1,3-diethyl-1H-1,3-benzodiazol-3-iumformate Example 38 (99%, 250 mg, 0.36 mmol) in dioxane (4 ml). Theresulting mixture was stirred at RT for 30 min then heated to 40° C. for45 min. MeCN (10 ml) was added then the reaction was heated at 40° C.for 45 min then concentrated in vacuo. The residue was suspended indioxane (4 ml) then HCl solution in dioxane (4.0 M, 1.6 ml, 6.4 mmol)was added. MeOH was added drop-wise until the suspension became ahomogeneous solution. The resulting solution was stirred at RT for 1 hthen concentrated in vacuo. The residue was suspended in MeCN (15 ml)with sonication then the solid was left to settle. The supernatant wasdecanted off with a pipette then the trituration process was repeated(×3). The residual solid was dried under vacuum then purified by flashcolumn chromatography on C18 (30 g). The column was eluted withMeCN:water+0.1% TFA using the following gradient (% MeCN, columnvolumes): 0%, 2 CV; 0-20%, 20 CV; 20-30%, 2 CV; 30-100%, 2 CV; 100%, 2CV. The desired fractions were combined and concentrated in vacuo. Theresidue was re-dissolved in water:MeCN (9:1) then lyophilised to affordthe product as an orange solid (190 mg, 68%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.55 (s, 1H), 9.73 (t, J=5.3 Hz, 1H),9.25-9.11 (m, 3H), 8.95 (d, J=10.0 Hz, 1H), 8.79-8.65 (m, 1H), 8.54 (s,1H), 8.21-8.15 (m, 2H), 7.80-6.80 (m, 2H), 6.42 (dd, J=3.8, 1.7 Hz, 1H),5.11 (d, J=5.3 Hz, 2H), 4.77-4.68 (m, 4H), 3.65 (q, J=6.2 Hz, 2H), 3.41(d, J=12.5 Hz, 3H), 3.25-3.15 (m, 2H), 2.94 (q, J=12.4 Hz, 2H), 2.20 (d,J=12.5 Hz, 2H), 1.81-1.68 (m, 2H), 1.49-1.38 (m, 6H).

LC/MS (System D): m/z (ESI⁺)=533 [M⁺], Rt=1.31 min, UV purity=99%.

Example 43—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-5-(2-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}ethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

A solution of2-(aminomethyl)-5-(2-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}ethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumdihydrochloride chloride, Intermediate 89 (70%, 1.53 g, 1.53 mmol),(3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl)-imidazol-1-yl-methanone,Intermediate 4 (734 mg, 3.22 mmol) and imidazole hydrochloride (336 g,3.22 mmol) in DMF (14 ml) was stirred at RT for 44 h. Additional(3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl)-imidazol-1-yl-methanone,Intermediate 4 (150 mg, 0.657 mmol) was added then the reaction was leftto stir for a further 72 h at RT. The reaction mixture was diluted withwater (30 ml) and filtered. The solid was rinsed through with water(2×10 ml). The filtrate was concentrated in vacuo to a brown/yellow oil.The crude material was purified by flash column chromatography on C18(60 g). The column was eluted with MeCN:water+0.1% TFA using thefollowing gradient (% MeCN, column volumes): 10%, 2 CV; 10-18%, 15 CV;18-25%, 2 CV; 25-30%, 1 CV; 30%, 1 CV; 30-36%, 1 CV; 36-40%, 1 CV;40-100%, 4 CV; 100%, 1 CV. The desired fractions were combined andlyophilised to afford a yellow solid (524 mg). A sample (120 mg) of thematerial thus obtained was further purified by HPLC (Method D). Thedesired fractions were combined and lyophilised to afford an orangesolid (54 mg). The material thus obtained was further purified by flashcolumn chromatography on C18 (60 g). The column was eluted withMeCN:water+0.1% TFA using the following gradient (% MeCN, columnvolumes): 0%, 7 CV; 0-20%, 17 CV; 20%, 3 CV; 20-100%, 3 CV; 100%, 1 CV.The desired fractions were combined and lyophilised to afford theproduct as an orange solid (14 mg, 1%).

¹H NMR (500 MHz, CD₃OD) δ 7.89 (d, J=9.1 Hz, 1H), 7.57 (d, J=1.9 Hz,1H), 7.43 (dd, J=9.1, 2.1 Hz, 1H), 7.39 (d, J=3.8 Hz, 1H), 6.44 (d,J=3.8 Hz, 1H), 5.09 (s, 2H), 4.76 (q, J=7.2 Hz, 4H), 4.57 (s (br), 2H),4.22 (s (br), 2H), 4.01-3.41 (m, 16H), 1.58-1.51 (m, 6H).

LC/MS (System D): m/z (ESI⁺)=751 [M⁺], Rt=1.42 min, UV purity=98%.

Example 44—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-5-(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

A mixture of2-(aminomethyl)-5-(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrochloride chloride, Intermediate 93 (80%, 200 mg, 0.24 mmol),2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 4 (95%, 120 mg, 0.50 mmol) and 1H-imidazole hydrochloride(1:1) (52 mg, 0.50 mmol) in DMF (2.5 ml) was stirred at RT for 64 h. Thereaction mixture was diluted with water (5 ml) then the solid wascollected by filtration and washed with water (2×5 ml). The combinedfiltrate was concentrated in vacuo to afford the crude product as ayellow/brown oil. The crude material was purified by flash columnchromatography on C18 (60 g). The column was eluted with MeCN:water+0.1%TFA using the following gradient (% MeCN, column volumes): 10%, 2 CV;10-19%, 11 CV; 19-25%, 3 CV; 25-100%, 2 CV; 100%, 1 CV. The desiredfractions were combined and lyophilised to afford the product as ayellow solid (40 mg, 15%).

¹H NMR (500 MHz, CD₃OD) δ 7.88 (d, J=9.2 Hz, 1H), 7.51 (d, J=2.1 Hz,1H), 7.44-7.38 (m, 2H), 6.46 (d, J=3.8 Hz, 1H), 5.10 (s, 2H), 4.78 (q,J=7.3 Hz, 4H), 4.38-4.30 (m, 2H), 4.25 (s (br), 2H), 3.86 (s, 2H),3.81-3.75 (m, 2H), 3.74-3.51 (m, 10H), 3.50-3.40 (m, 2H), 2.38 (s (br),2H), 1.56 (t, J=7.1 Hz, 6H).

LC/MS (System D): m/z (ESI⁺)=765 [M⁺], Rt=1.36 min, UV purity=95%.

Example 45—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-5-(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

Step 1: A mixture of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-aminopropyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrochloride chloride, Example 29 (350 mg, 0.55 mmol),4,6-O-benzylidene-D-glucopyranose (571 mg, 2.13 mmol) and AcOH (122 μl,2.13 mmol) in MeOH (30 ml) was stirred at RT for 0.5 h. NaCNBH₃ (134 mg,2.13 mmol) was added. The resulting mixture was stirred at RT for 16 h.The reaction mixture was re-charged with4,6-O-benzylidene-D-glucopyranose (591 mg, 2.20 mmol) and NaCNBH₃ (138mg, 2.20 mmol) then left to stir at RT for a further 24 h. The reactionmixture was re-charged with 4,6-O-benzylidene-D-glucopyranose (295 mg,1.10 mmol) and NaCNBH₃ (69 mg, 1.1 mmol) then left to stir at RT for afurther 96 h. Saturated aqueous NaHCO₃ (30 ml) solution was addeddropwise over 5 min then the resultant suspension was sonicated andfiltered. The solid collected was washed with water (20 ml) then driedunder vacuum. The crude solid thus obtained was purified by flash columnchromatography on C18 (60 g). The column was eluted with MeCN:water,sing the following gradient (% MeCN, column volumes): 0%, 2 CV; 0-100%,12 CV; 100-5%, 2 CV. The column was then further eluted with MeCN:water,+0.1% TFA using the following gradient (% MeCN, column volumes): 0%, 2CV; 0-50%, 8 CV; 50%, 3 CV; 50-100%, 5 CV. The desired fractions werecombined and concentrated in vacuo to afford the protected product as ayellow oil (85 mg). Step 2: 2.0 M aqueous HCl solution (0.53 ml) wasadded to a solution of the intermediate from Step 1,2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-5-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide (80%, 70 mg, 0.053 mmol) in EtOH:water (1:1, 2 ml). The reactionmixture was stirred for 4 h at RT. The reaction mixture was concentratedin vacuo. The crude material was purified by preparative HPLC (MethodE). The desired fractions were combined and lyophilised to afford theproduct as yellow solid (20 mg, 38% over 2 steps).

¹H NMR (500 MHz, CD₃OD) δ 7.93 (d, J=8.6 Hz, 1H), 7.89 (s, 1H), 7.65(dd, J=8.6, 0.8 Hz, 1H), 7.41 (d, J=3.8 Hz, 1H), 6.46 (d, J=3.8 Hz, 1H),5.17-5.09 (m, 2H), 4.82-4.76 (m, 4H), 4.21-4.10 (m, 2H), 3.84 (dd,J=4.5, 1.6 Hz, 2H), 3.79 (dd, J=11.0, 3.4 Hz, 2H), 3.75-3.62 (m, 6H),3.52-3.37 (m, 4H), 3.10-2.92 (m, 2H), 2.22 (s, 2H), 1.57 (q, J=7.1 Hz,6H), 1.32 (d, J=7.3 Hz, 2H).

LC/MS (System C): m/z (ESI⁺)=749 [M⁺], Rt=1.43 min, UV purity=98%.

Example 46—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-{1-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]piperidin-4-yl}-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

2.0 M aqueous HCl solution (4.7 ml, 9.4 mmol) was added to a solution of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{1-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]piperidin-4-yl}-1,3-diethyl-1H-1,3-benzodiazol-3-iumiodide, Intermediate 99 (390 mg, 0.470 mmol) in water (3 ml). Theresulting solution was sonicated for 5 min then stirred at RT for 1.5 h.The reaction mixture was concentrated in vacuo then purified by flashcolumn chromatography on C18 (30 g). The column was eluted withMeCN:water+0.1% TFA using the following gradient (% MeCN, columnvolumes): 0%, 2 CV; 0-6%, 4 CV; 6-13%, 4 CV; 13-25%, 8 CV; 25-42%, 2 CV;42-52%, 1 CV; 52-100%, 1 CV; 100% 2 CV. The desired fractions werecombined and lyophilised to afford the product as a yellow solid (120mg, 30%).

¹H NMR (250 MHz, 353 K, 5% D₂O in DMSO-d₆) δ 7.97 (d, J=8.7 Hz, 1H),7.83 (s, 1H), 7.59 (d, J=8.4 Hz, 1H), 7.44 (d, J=3.8 Hz, 1H), 6.40 (d,J=3.8 Hz, 1H), 5.08 (s, 2H), 4.75-4.60 (m, 4H), 4.18-4.10 (m, 1H),3.77-3.41 (m, 8H), 3.36-3.00 (m, 4H+HDO), 2.11 (s, 4H), 1.44 (dt, J=7.1,3.5 Hz, 6H).

LC/MS (System C): m/z (ESI⁺)=611 [M⁺], Rt=0.91 min, UV purity=100%.

Example 47—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-(1-{1-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]piperidin-4-yl}-1H-pyrazol-4-yl)-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

Step 1: A solution of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]-1H-1,3-benzodiazol-3-iumhydrochloride iodide, Example 31 (360 mg, 0.53 mmol) in MeOH (20 ml),4,6-O-benzylidene-D-glucopyranose (417 mg, 1.55 mmol) and AcOH (89 μl,1.56 mmol) was stirred at RT for 20 min. NaCNBH₃ (98 mg, 1.56 mmol) wasadded then the resulting mixture was stirred at RT for 48 h. Thereaction was recharged with 4,6-O-benzylidene-D-glucopyranose (142 mg,0.53 mmol) and AcOH (81 μl, 0.53 mmol) then the reaction was stirred atRT for a further 4 h. Saturated aqueous NaHCO₃ (40 ml) solution wasadded dropwise over 5 min then the resultant suspension was filtered.The solid collected was washed with water (40 ml) then dried undervacuum to afford the protected intermediate as a yellow solid. Step 2:Aqueous HCl solution (2.0 M, 10 ml, 20 mmol) was added to a solution ofthe Intermediate from Step 1,2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(1-{1-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]piperidin-4-yl}-1H-pyrazol-4-yl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumchloride (70%, 275 mg, 0.251 mmol) in water (5 ml). The reaction wasstirred at RT for 5 min then THF (5 ml) was added. The reaction mixturewas concentrated in vacuo then the crude material was purified by flashcolumn chromatography on C18 (30 g). The column was eluted withMeCN:water using the following gradient (% MeCN, column volumes): 0%, 3CV; 2-10%, 2 CV; 10-21%, 3 CV; 21-42%, 1 CV; 42-50%, 1 CV. The columnwas then eluted with MeCN:water+0.1% formic acid using the followinggradient (% MeCN, column volumes): 0%, 2 CV; 0-10%, 4 CV; 10-29%, 1 CV;29-57%, 1 CV; 29-57%, 1 CV; 100%, 2 CV. The desired fractions werecombined and concentrated in vacuo to afford a black gum (280 mg). Thematerial thus obtained was further purified by preparative HPLC (MethodE). The desired fractions were combined then lyophilised to afford theproduct as a yellow solid (18 mg, 8%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.53 (s, 1H), 9.72 (t, J=5.3 Hz, 1H), 9.23(s, 1H), 8.57-8.43 (m, 1H), 8.45 (s, 0.75H), 8.25 (s, 1H), 8.18 (d,J=12.1 Hz, 1H), 8.06 (dd, J=8.7, 4.8 Hz, 1H), 7.94 (dd, J=8.6, 1.2 Hz,1H), 7.52 (dd, J=3.7, 2.5 Hz, 1H), 7.22 (s, 2H), 6.42 (dd, J=3.8, 1.7Hz, 1H), 5.50 (s, 1H), 5.07 (d, J=5.2 Hz, 2H), 4.81 (d, J=45.4 Hz, 1H),4.69 (q, J=6.8 Hz, 4H), 4.61 (s, 2H), 4.54-4.38 (m, 2H), 4.09-3.95 (m,1H), 3.73-3.57 (m, 4H), 3.56-3.39 (m, 4H), 3.28-3.13 (m, 5H), 2.34-2.20(m, 2H), 1.43 (dt, J=10.8, 7.2 Hz, 6H).

LC/MS (System C): m/z (ESI⁺)=677 [M⁺], Rt=1.20 min, UV purity=100%.

Example 48—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)carbamoyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

A solution of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (200 mg, 0.410 mmol) and CDI (100 mg, 0.614 mmol) inDMF (2 ml) was stirred at RT for 4 h. The resulting solution was addedto(2R,3R,4R,5S)-6-[(3-aminopropyl)[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino]hexane-1,2,3,4,5-pentoldihydrochloride, Intermediate 104 (234 mg, 0.490 mmol) and rinsed intothe reaction flask with DMF (2×1 ml). The resulting solution was stirredat RT for 18 h. The reaction mixture was concentrated in vacuo then thecrude material was purified by flash column chromatography on C18 (30g). The column was eluted with MeCN:water+0.1% TFA using the followinggradient (% MeCN, column volumes): 5%, 2 CV; 5-25%, 11 CV; 25%, 2 CV;25-34%, 1 CV; 34-100%, 2 CV; 100%, 1 CV. The desired fractions werecombined and lyophilised to afford the product as a yellow solid (160mg, 38%).

δ (ppm): ¹H NMR (500 MHz, CD₃OD) δ 8.47 (s, 1H), 8.18 (dd, J=8.8, 1.4Hz, 1H), 8.08 (d, J=8.8 Hz, 1H), 7.39 (d, J=3.8 Hz, 1H), 6.44 (d, J=3.8Hz, 1H), 5.19-5.10 (m, 2H), 4.94-4.74 (m, 4H+HDO), 4.26-4.15 (m, 2H),3.88-3.81 (m, 2H), 3.81-3.73 (m, 2H), 3.73-3.38 (m, 14H), 2.24-2.10 (m,2H), 1.67-1.51 (m, 6H).

LC/MS (System D): m/z (ESI⁺)=792 [M⁺], Rt=1.24 min, UV purity=100%.

Example 49—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-({1-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]piperidin-4-yl}carbamoyl)-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

A mixture of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (150 mg, 0.31 mmol) and CDI (75 mg, 0.46 mmol) inDMF (8 ml) was stirred for 1 h at RT then(2R,3R,4R,5S)-6-(4-aminopiperidin-1-yl)hexane-1,2,3,4,5-pentoldihydrochloride, Intermediate 109 (140 mg, 0.42 mmol) was added. Thereaction was left to stir at RT for 64 h then concentrated in vacuo. Thecrude material was purified by flash column chromatography on C18 (30g). The column was eluted with MeCN:water+0.1% TFA using the followinggradient (% MeCN, column volumes): 0%, 2 CV; 0-8%, 6 CV; 8-9%, 1 CV;9-20%, 5 CV; 20-25%, 2 CV; 25-44%, 3 CV; 44-51%, 1 CV; 51-100%, 1 CV;100%, 1 CV. The desired fractions were combined and lyophilised toafford the product as a yellow solid (46 mg, 17%).

¹H NMR (500 MHz, 5% D₂O in DMSO-d₆) δ 8.37 (s, 1H), 8.08 (s, 2H), 7.46(d, J=3.8 Hz, 1H), 6.43 (d, J=3.8 Hz, 1H), 5.06 (s, 2H), 4.75-4.61 (m,4H), 4.19-3.97 (m, 2H), 3.68-−3.30 (m, 8H), 3.20-2.99 (m, 3H) (m, 3H),2.19-1.74 (m, 4H), 1.40 (dt, J=15.9, 7.4 Hz, 6H).

LC/MS (System D): m/z (ESI⁺)=654 [M⁺], Rt=1.38 min, UV purity=100%.

Example 50—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

A solution of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (210 mg, 0.43 mmol) and CDI (105 mg, 0.65 mmol) inDMF (2 ml) was stirred at RT for 1.5 h. The reaction mixture was addedto(2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl](piperidin-4-yl)amino}hexane-1,2,3,4,5-pentoldihydrochloride, Intermediate 112 (260 mg, 0.52 mmol). The reactionmixture was left to stir at RT for 16 h then concentrated in vacuo. Thecrude material was purified by flash column chromatography on C18 (30g). The column was eluted with MeCN:water+0.1% TFA using the followinggradient (% MeCN, column volumes): 5%, 2 CV; 5-12%, 6 CV; 12%, 3 CV;12-20%, 6 CV; 20-51%, 3 CV; 25-93%, 2 CV. The desired fractions werecombined and lyophilised to afford the product as a yellow solid (114mg, 25%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.54 (s, 1H), 9.74 (t, J=5.3 Hz, 1H),8.34-8.07 (m, 3H), 7.73 (d, J=9.3 Hz, 1H), 7.57-7.48 (m, 1H), 7.32-7.06(m, 2H), 6.46-6.38 (m, 1H), 5.64-5.44 (m, 2H), 5.14-5.02 (m, 2H),4.94-4.33 (m, 12H), 4.12-3.93 (m, 2H), 3.90-3.78 (m, 1H), 3.77-3.69 (m,2H), 3.33 (15H+HDO), 2.95-2.74 (m, 1H), 2.27-1.55 (m, 4H), 1.49-1.36 (m,6H).

LC/MS (System D): m/z (ESI⁺)=818 [M⁺], Rt=1.33 min, UV purity=100%.

Example 51—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{[2-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidin-1-yl)ethyl]carbamoyl}-1,3-diethyl-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

A solution of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (210 mg, 0.43 mmol) and CDI (98 mg, 0.60 mmol) inDMF (5 ml) was stirred at RT for 45 min. The resultant solution wasadded to(2R,3R,4R,5S)-6-{[1-(2-aminoethyl)piperidin-4-yl][(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}hexane-1,2,3,4,5-pentoltrihydrochloride, Intermediate 116 (88%, 500 mg, 0.76 mmol) and rinsedinto the flask with DMF (0.5 ml). The resultant reaction mixture wasstirred at RT for 64 h. The reaction mixture was concentrated in vacuothen purified by flash column chromatography on C18 (30 g). The columnwas eluted with MeCN:water+0.1% TFA using the following gradient (%MeCN, column volumes): 0%, 2 CV; 0-20%, 20 CV; 20%, 2 CV; 20-100%, 2 CV;100% 2 CV. The desired fractions were combined and lyophilised to affordthe product as a yellow solid (216 mg, 46%).

¹H NMR (500 MHz, CD₃OD) δ 8.52 (s, 1H), 8.24 (d, J=8.7 Hz, 1H), 8.12 (d,J=8.8 Hz, 1H), 7.43 (d, J=3.8 Hz, 1H), 6.48 (d, J=3.8 Hz, 1H), 5.19 (s,2H), 4.93-4.84 (m, 4H+HDO), 4.22 (s, 2H), 4.10-3.63 (m, 16H), 3.55-3.38(m, 5H), 3.22-3.05 (m, 2H), 2.50-2.09 (m, 4H), 1.68-1.56 (m, 6H).

LC/MS (System D): m/z (ESI⁺)=431.5 [(M⁺)⁺H+], Rt=1.31 min, UVpurity=100%.

Example 52—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-5-[4-({bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}methyl)piperidine-1-carbonyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

A solution of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (196 mg, 0.402 mmol) and CDI (98 mg, 0.60 mmol) inDMF (1.5 ml) was stirred at RT for 3.5 h.(2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-Pentahydroxyhexyl][(piperidin-4-yl)methyl]amino}hexane-1,2,3,4,5-pentoldihydrochloride, Intermediate 121 (93%, 248 mg, 0.448 mmol) was addedthen the reaction was left to stir at RT for 16 h. The reaction mixturewas purified by flash column chromatography on C18 (30 g). The columnwas eluted with MeCN:H₂O+0.1% TFA using the following gradient (% MeCN,column volumes): 5%, 1.5 CV; 5-25%, 10.5 CV; 25%, 2 CV; 25-34%, 1 CV;34-100%, 2 CV; 100% 2 CV. The desired fractions were combined andlyophilised to afford the product as a yellow solid (72 mg, 17%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.55 (s, 1H), 9.75 (t, J=5.3 Hz, 1H),8.20-8.09 (m, 2H), 7.83 (s, 1H), 7.73-7.62 (m, 1H), 7.58-7.48 (m, 1H),7.21 (s, 2H), 6.48-6.38 (m, 1H), 5.73-5.48 (m, 2H), 5.18-5.01 (m, 2H),4.97-4.40 (m, 13H), 4.09-3.87 (m, 2H), 3.77-3.66 (m, 2H), 3.65-3.06 (m,16H), 2.97-2.73 (m, 1H), 2.27-2.08 (m, 1H), 2.04-1.52 (m, 2H), 1.52-1.36(m, 6H), 1.35-1.13 (m, 2H).

LC/MS (System D): m/z (ESI⁺)=832 [M⁺], R_(t)=1.33 min, UV purity=99%.

Example 53—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3R)-3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}pyrrolidine-1-carbonyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

A suspension of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (200 mg, 0.410 mmol) and CDI (100 mg, 0.61 mmol) inDMF (2 ml) was stirred at RT for 1 h.(2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl][(3R)-pyrrolidin-3-yl]amino}hexane-1,2,3,4,5-pentoldihydrochloride, Intermediate 123 (83%, 289 mg, 0.492 mmol) was addedthen the reaction was left to stir at RT for 64 h. The reaction mixturewas concentrated under a stream of nitrogen, then the crude material waspurified by flash column chromatography on C18 (12 g). The column waseluted with MeCN:H₂O+TFA using the following gradient (% MeCN, columnvolumes): 2%, 2 CV; 2-25%, 15 CV; 25-100%, 2 CV. The desired fractionswere combined and concentrated in vacuo then lyophilised to afford ayellow solid (140 mg). The material thus obtained was further purifiedby flash column chromatography on C18 (12 g). The column was eluted withMeCN:H₂O+TFA using the following gradient (% MeCN, column volumes): 2%,1 CV; 2-20%, 10 CV; 20-100%, 2 CV. The desired fractions were combinedand concentrated in vacuo then lyophilised to afford a yellow solid (65mg, 15%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.54 (s, 1H), 9.74 (t, J=4.7 Hz, 1H),9.33-8.80 (m, 1H), 8.29-8.21 (m, 1H), 8.20-8.11 (m, 1H), 7.85-7.76 (m,1H), 7.53 (dd, J=3.7, 2.6 Hz, 1H), 7.39-7.05 (m, 2H), 6.42 (dd, J=3.8,1.8 Hz, 1H), 5.62-5.21 (m, 2H), 5.09 (d, J=4.9 Hz, 2H), 4.94-3.45 (m,33H), 2.34-2.04 (m, 2H), 1.46-1.35 (m, 6H).

LC/MS (System D): m/z (ESI⁺)=804 [M⁺], R_(t)=1.27 min, UV purity=98%.

Example 54—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3S)-3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}pyrrolidine-1-carbonyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

A suspension of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (200 mg, 0.410 mmol) and CDI (100 mg, 0.61 mmol) inDMF (2 ml) was stirred at RT for 1 h.(2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-Pentahydroxyhexyl][(3S)-pyrrolidin-3-yl]amino}hexane-1,2,3,4,5-pentoldihydrochloride, Intermediate 125 (240 mg, 0.492 mmol) was added thenthe reaction was left to stir at RT for 16 h. The reaction mixture wasconcentrated under a stream of nitrogen, then the crude material waspurified by flash column chromatography on C18 (30 g). The column waseluted with MeCN:H₂O+TFA using the following gradient (% MeCN, columnvolumes): 2%, 2 CV; 2-20%, 10 CV; 20-100%, 2 CV. The desired fractionswere combined and concentrated in vacuo then lyophilised to afford ayellow solid (140 mg). The material thus obtained was further purifiedby flash column chromatography on C18 (12 g). The column was eluted withMeCN:H₂O+TFA using the following gradient (% MeCN, column volumes): 2%,1 CV; 2-20%, 10 CV; 20-100%, 2 CV. The desired fractions were combinedand concentrated in vacuo then lyophilised to afford a yellow solid (232mg, 54%).

¹H NMR (500 MHz, DMSO-d₆+D₂O) b 9.79-9.68 (m, 1H), 8.18-8.11 (m, 1H),8.11-8.04 (m, 1H), 7.76 (d, J=8.8 Hz, 1H), 7.46 (d, J=3.8 Hz, 1H), 6.43(d, J=3.8 Hz, 1H), 5.12-4.97 (m, 2H), 4.77-4.59 (m, 4H), 4.41-4.19 (m,1H), 4.13-3.95 (m, 2H), 3.79-3.20 (m, 17H), 2.47-2.34 (m, 2H), 2.29-2.12(m, 1H), 1.45-1.32 (m, 6H).

LC/MS (System D): m/z (ESI⁺)=804 [M⁺], R_(t)=1.27 min, UV purity=99%.

Example 55—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-{[(1r,4r)-4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}cyclohexyl]carbamoyl}-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

A suspension of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (400 mg, 0.819 mmol) and CDI (199 mg, 1.23 mmol) inDMF (4 ml) was stirred at RT for 1 h.(2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-Pentahydroxyhexyl][(1r,4r)-4-aminocyclohexyl]amino}hexane-1,2,3,4,5-pentoldihydrochloride, Intermediate 127 (90%, 550 mg, 0.960 mmol) was addedthen the reaction was left to stir at RT for 16 h. The reaction mixturewas concentrated under a stream of nitrogen, then the crude material waspurified by flash column chromatography on C18 (30 g). The column waseluted with MeCN:H₂O+TFA using the following gradient (% MeCN, columnvolumes): 2%, 2 CV; 2-25%, 15 CV; 25-100%, 2 CV. The desired fractionswere combined and concentrated in vacuo to afford a yellow/orange solid(292 mg). The material thus obtained was dissolved in water (3 ml) thenan aliquot (1 ml) was purified by column chromatography on C4 (12 g).The column was eluted with MeCN:H₂O+TFA using the following gradient (%MeCN, column volumes): 5%, 2 CV; 5-22%, 10 CV; 22-100%, 2 CV, 100%, 2CV. A second aliquot (1 ml) was purified by column chromatography on acyano column (13 g). The column was eluted with MeCN:H₂O+TFA using thefollowing gradient (% MeCN, column volumes): 2%, 2 CV; 2-4%, 2 CV; 4-8%,2 CV; 8-100%, 2 CV, 100%, 2 CV. An further aliquot (1 ml) was purifiedby column chromatography on C18 (12 g). The column was eluted withMeCN:H₂O+TFA using the following gradient (% MeCN, column volumes): 2%,2 CV; 2-15%, 11 CV; 15-20%, 1 CV; 20-100%, 2 CV, 100%, 2 CV. The desiredfractions from the three columns were combined and concentrated in vacuothen lyophilised to afford two batches of yellow solid (77 mg and 163mg). Both batches of solid thus obtained were further purified by columnchromatography on C18 (12 g). The column was eluted with MeCN:H₂O+TFAusing the following gradient (% MeCN, column volumes): 2%, 2 CV; 2-15%,10 CV; 15-100%, 2 CV, 100%, 2 CV. The desired fractions from bothcolumns were combined and concentrated in vacuo then lyophilised toafford a yellow solid (53 mg). The material thus obtained was furtherpurified by HPLC purification under the following conditions: stationaryphase: XSelect CSH C18 30×100 mm, 5 μm; detection UV 220 nm; mobilephase A: water+0.1% TFA; B: MeCN+0.1% TFA; gradient: 1-15% solvent Bover 18 min; flowrate: 42 ml/min. The desired fractions were combinedand concentrated in vacuo then lyophilised to afford the product as ayellow solid (26 mg, 3.0%).

¹H NMR (500 MHz, DMSO-d₆+D₂O) b 9.73 (t, J=5.4 Hz, 1H), 8.65-8.54 (m,1H), 8.40 (s, 1H), 8.10 (s, 2H), 7.49 (d, J=3.8 Hz, 1H), 6.42 (d, J=3.8Hz, 1H), 5.07 (d, J=4.5 Hz, 2H), 4.79-4.62 (m, 4H), 4.29-3.76 (m, 6H),3.56-3.10 (m, 11H), 2.23-1.56 (m, 7H), 1.50-1.32 (m, 8H).

LC/MS (System D): m/z (ESI⁺)=832 [M⁺], R_(t)=1.38 min, UV purity=99%.

Example 56—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-{[(1s,4s)-4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}cyclohexyl]carbamoyl}-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

A suspension of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (185 mg, 0.379 mmol) and CDI (92 mg, 0.57 mmol) inDMF (1.5 ml) was stirred at RT for 2 h. The reaction mixture was addedto2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-Pentahydroxyhexyl][(1s,4s)-4-aminocyclohexyl]amino}hexane-1,2,3,4,5-pentol,Intermediate 132 (210 mg, 0.475 mmol) and rinsed in DMF (1 ml). Thereaction was left to stir at RT for 16 h. The reaction mixture wasconcentrated in vacuo then the crude material was purified by flashcolumn chromatography on C18 (30 g). The column was eluted withMeCN:H₂O+TFA using the following gradient (% MeCN, column volumes): 2%,2 CV; 2-20%, 15 CV; 20-100%, 2 CV; 100%, 2 CV. The desired fractionswere combined and lyophilised to afford a yellow solid (42 mg). A sample(31 mg) of the material thus obtained was further purified by flashcolumn chromatography on C18 (12 g). The column was eluted withMeCN:H₂O+TFA using the following gradient (% MeCN, column volumes): 2%,2 CV; 2-20%, 10 CV; 20-100%, 2 CV; 100%, 2 CV. The desired fractionswere combined and concentrated in vacuo then lyophilised to afford theproduct as a yellow solid (30 mg, 7.4%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.59-11.50 (m, 1H), 9.74 (t, J=5.3 Hz, 1H),8.40 (s, 1H), 8.16-8.11 (m, 1H), 7.93-7.84 (m, 1H), 7.53 (dd, J=3.7, 2.6Hz, 1H), 7.35-7.02 (m, 2H), 6.45-6.40 (m, 1H), 5.77-5.47 (m, 2H),5.19-5.07 (m, 2H), 5.01-3.68 (m, 25H), 3.26-3.12 (m, 4H), 2.14-1.36 (m,16H).

LC/MS (System D): m/z (ESI⁺)=832 [M⁺], R_(t)=1.38 min, UV purity=99%.

Example 57—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)(methyl)carbamoyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

A suspension of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (200 mg, 0.410 mmol) and CDI (100 mg, 0.61 mmol) inDMF (2 ml) was stirred at RT for 1 h.(2R,3R,4R,5S)-6-{[3-(Methylamino)propyl][(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}hexane-1,2,3,4,5-pentoldihydrochloride, Intermediate 134 (241 mg, 0.492 mmol) was added thenthe reaction was left to stir at RT for 16 h. The reaction mixture wasconcentrated under a stream of nitrogen, then the crude material waspurified by flash column chromatography on C18 (30 g). The column waseluted with MeCN:H₂O+TFA using the following gradient (% MeCN, columnvolumes): 2%, 2 CV; 2-20%, 10 CV; 20-100%, 2 CV; 100%, 2 CV. The desiredfractions were combined and concentrated in vacuo then lyophilised toafford the product as a yellow solid (35 mg, 8.0%).

¹H NMR (500 MHz, DMSO-d₆+D₂O) b 9.79-9.71 (m, 1H), 8.13-8.05 (m, 2H),7.74-7.59 (m, 1H), 7.49 (d, J=3.8 Hz, 1H), 6.43 (d, J=3.8 Hz, 1H),5.13-4.98 (m, 2H), 4.80-4.63 (m, 4H), 4.09-3.81 (m, 2H), 3.66-3.14 (m,17H), 3.05-2.85 (m, 3H), 2.58-2.54 (m, 1H), 2.13-1.83 (m, 2H), 1.47-1.33(m, 6H).

LC/MS (System D): m/z (ESI⁺)=806 [M⁺], R_(t)=1.30 min, UV purity=97%.

Example 58—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(2-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}ethyl)carbamoyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

A suspension of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (200 mg, 0.410 mmol) and CDI (100 mg, 0.61 mmol) inDMF (3 ml) was stirred at RT for 1.5 h. The resultant solution was addedto(2R,3R,4R,5S)-6-[(2-aminoethyl)[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino]hexane-1,2,3,4,5-pentoldihydrochloride, Intermediate 136 (94%, 227 mg, 0.462 mmol) and rinsedinto the flask with DMF (1.5 ml). The reaction was stirred at RT for 16h then concentrated in vacuo, then the crude material was purified byflash column chromatography on C18 (30 g). The column was eluted withMeCN:H₂O+TFA using the following gradient (% MeCN, column volumes): 5%,2 CV; 5-25%, 10.5 CV; 25%, 2 CV; 25-34%, 1 CV; 34-100%, 2 CV; 100%, 2CV. The desired fractions were combined and concentrated in vacuo thenlyophilised to afford the product as a yellow solid (101 mg, 24%).

¹H NMR (500 MHz, CD₃OD) δ 8.51-8.43 (m, 1H), 8.19 (dd, J=8.7, 1.3 Hz,1H), 8.09 (d, J=8.8 Hz, 1H), 7.39 (d, J=3.8 Hz, 1H), 6.45 (d, J=3.8 Hz,1H), 5.19-5.08 (m, 2H), 4.86-4.75 (m, 4H), 4.29-4.19 (m, 2H), 3.99-3.47(m, 18H), 1.67-1.49 (m, 6H).

LC/MS (System D): m/z (ESI⁺)=778 [M⁺], R_(t)=1.32 min, UV purity=98%.

Example 59—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-5-{[(14S,15R,16R,17R)-14,15,16,17,18-pentahydroxy-12-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]-3,6,9-trioxa-12-azaoctadecan-1-yl]carbamoyl}-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

CDI (99 mg, 0.61 mmol) was added to a suspension of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (217 mg, 0.444 mmol) in DMF (1.5 ml). The reactionwas left to stir at RT for 4 h then added to(14S,15R,16R,17R)-1-amino-12-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]-3,6,9-trioxa-12-azaoctadecane-14,15,16,17,18-pentoldihydrochloride, Intermediate 141 (96%, 289 mg, 0.467 mmol) and rinsedin with DMF (1 ml). The reaction was stirred at RT for 16 h. The crudematerial was purified by flash column chromatography on C18 (30 g). Thecolumn was eluted with MeCN:H₂O+TFA using the following gradient (%MeCN, column volumes): 2%, 2 CV; 2-6%, 3 CV; 6%, 1 CV; 6-13%, 6 CV; 13%,5 CV; 13-20%, 5 CV; 20%, 2 CV; 20-100%, 2 CV; 100%, 1 CV. The desiredfractions were combined and lyophilised to afford a yellow solid (101mg). The material thus obtained was further purified by flash columnchromatography on C18 (12 g). The column was eluted with MeCN:H₂O+TFAusing the following gradient (% MeCN, column volumes): 2%, 1.5 CV;2-10%, 3 CV; 10-12%, 1 CV; 12%, 6 CV. The desired fractions werecombined and lyophilised to afford the product as a yellow solid (77 mg,15%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.58-11.46 (m, 1H), 9.73 (t, J=5.2 Hz, 1H),8.85 (t, J=5.6 Hz, 1H), 8.50 (s, 1H), 8.29-8.03 (m, 3H), 7.52 (dd,J=3.7, 2.6 Hz, 1H), 7.33-7.12 (m, 2H), 6.42 (dd, J=3.8, 1.7 Hz, 1H),5.50-5.28 (m, 1H), 5.10 (d, J=5.2 Hz, 2H), 4.90-4.62 (m, 5H), 4.61-4.34(m, 5H), 4.03-3.90 (m, 2H), 3.81-3.33 (m, 33H), 1.50-1.34 (m, 6H).

LC/MS (System D): m/z (ESI⁺)=910 [M⁺], R_(t)=1.50 min, UV purity=99%.

Example 60—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-({2-[4′-(2-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}ethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamoyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

CDI (93 mg, 0.57 mmol) was added to a suspension of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (187 mg, 0.38 mmol) in DMF (1.5 ml). The reactionwas left to stir at RT for 5 h then(2R,3R,4R,5S)-6-({2-[4′-(2-aminoethyl)-[1,1′-biphenyl]-4-yl]ethyl}[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino)hexane-1,2,3,4,5-pentoldihydrochloride, Intermediate 146 (94%, 459 mg, 0.672 mmol) and DMF (0.5ml) were added. The reaction was left to stir at RT for a further 17 h.The crude material was purified by flash column chromatography on C18(30 g). The column was eluted with MeCN:H₂O+TFA using the followinggradient (% MeCN, column volumes): 2%, 2 CV; 2-9%, 6 CV; 9%, 3 CV;9-16%, 6 CV; 16-17%, 1 CV; 17%, 16 CV. The desired fractions werecombined and lyophilised to afford the product as a yellow solid (156mg, 33%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.61-11.47 (m, 1H), 9.73 (t, J=5.4 Hz, 1H),8.92 (t, J=5.4 Hz, 1H), 8.82-8.65 (m, 1H), 8.49 (s, 1H), 8.22-8.09 (m,2H), 7.66-7.58 (m, 4H), 7.52 (dd, J=3.7, 2.5 Hz, 1H), 7.41-7.03 (m, 6H),6.42 (dd, J=3.8, 1.8 Hz, 1H), 5.70-5.30 (m, 2H), 5.17-5.00 (m, 3H),4.77-4.67 (m, 5H), 4.14-4.00 (m, 4H), 3.75-3.25 (m, 22H+HDO), 3.10-3.01(m, 2H), 2.94 (t, J=7.3 Hz, 2H), 1.49-1.38 (m, 6H).

LC/MS (System D): m/z (ESI⁺)=480 [(M⁺H)²⁺], R_(t)=1.50 min, UVpurity=99%.

Example 61—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{[(3S)-3-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)amino]-3-carbamoylpropyl]carbamoyl}-1,3-diethyl-1H-1,3-benzodiazol-3-iumbis(trifluoroacetic acid) trifluoroacetate

A suspension of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (200 mg, 0.410 mmol) and CDI (100 mg, 0.614 mmol) inDMF (2 ml) was stirred at RT for 1.5 h then added to(2S)-4-amino-2-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)amino]butanamidetrihydrochloride, Intermediate 154 (91%, 323 mg, 0.480 mmol) and rinsedin with DMF (1 ml). The reaction was stirred at RT for 16 h thenconcentrated in vacuo. The crude material was purified by flash columnchromatography on C18 (30 g). The column was eluted with MeCN:H₂O+TFAusing the following gradient (% MeCN, column volumes): 5%, 2 CV; 5-25%,10.5 CV; 25%, 2 CV; 25-34%, 1 CV; 34-100%, 1 CV; 100%, 2 CV. The desiredfractions were combined, concentrated in vacuo then lyophilised toafford the product as a yellow solid (95 mg, 18%).

¹H NMR (500 MHz, CD₃OD) δ 8.53-8.45 (m, 1H), 8.21 (dd, J=8.8, 1.4 Hz,1H), 8.10 (d, J=8.8 Hz, 1H), 7.40 (d, J=3.8 Hz, 1H), 6.45 (d, J=3.8 Hz,1H), 5.15 (s, 2H), 4.86-4.77 (m, 4H), 4.26-4.17 (m, 2H), 4.02-3.97 (m,1H), 3.89-3.84 (m, 2H), 3.80-3.74 (m, 2H), 3.73-3.38 (m, 14H), 3.24-3.11(m, 2H), 2.33-2.21 (m, 4H), 1.65-1.53 (m, 6H).

LC/MS (System D): m/z (ESI⁺)=447 [(M⁺H)²⁺], R_(t)=1.32 min, UVpurity=95%.

Example 62—Synthesis of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{[4-(4-{3-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)amino]-3-carbamoylpropyl}phenyl)butyl]carbamoyl}-1,3-diethyl-1H-1,3-benzodiazol-3-iumbis(trifluoroacetic acid) trifluoroacetate

CDI (30 mg, 0.19 mmol) was added to a suspension of2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 34 (61 mg, 0.12 mmol) in DMF (1 ml). The reaction wasleft to stir at RT for 1.5 h then more CDI (4 mg, 0.02 mmol) was added.The reaction was left to stir at RT for a further 1 h then added to asuspension of4-[4-(4-aminobutyl)phenyl]-2-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)amino]butanamidetrihydrochloride, Intermediate 156 (88%, 106 mg, 0.125 mmol) in DMF (1ml). Further DMF (1 ml) was used to rinse the reaction mixture into theflask. The reaction was left to stir at RT for 16 h then concentrated invacuo. The crude material was purified by flash column chromatography onC18 (12 g). The column was eluted with MeCN:H₂O+TFA using the followinggradient (% MeCN, column volumes): 2%, 4 CV; 2-20%, 30 CV; 20-100%, 4CV; 100%, 4 CV. The desired fractions were combined then lyophilised toafford the product as a yellow solid (11 mg, 6.2%).

¹H NMR (500 MHz, D₂O) δ 8.28-8.24 (m, 1H), 8.04-7.96 (m, 2H), 7.54 (d,J=3.9 Hz, 1H), 7.28 (d, J=8.1 Hz, 2H), 7.23 (d, J=8.1 Hz, 2H), 6.56 (d,J=3.9 Hz, 1H), 5.23 (s, 2H), 4.77-4.71 (m, 4H), 4.29-4.22 (m, 2H),4.04-3.97 (m, 1H), 3.89-3.83 (m, 2H), 3.87-3.80 (m, 2H), 3.82-3.74 (m,2H), 3.71-3.63 (m, 4H), 3.54-3.28 (m, 8H), 3.24-3.09 (m, 2H), 2.78-2.63(m, 4H), 2.33-2.14 (m, 4H), 1.77-1.64 (m, 4H), 1.57-1.48 (m, 6H).

LC/MS (System D): m/z (ESI⁺)=513 [(M⁺H)²⁺], R_(t)=1.32 min, UVpurity=95%.

Example 63—Synthesis of2-[({3-amino-7-chloro-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumchloride

A suspension of2-(aminomethyl)-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrochloride chloride, Intermediate 159 (84%, 257 mg, 0.674 mmol) and7-chloro-2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 16 (85%, 248 mg, 0.803 mmol) in DMF (2.5 ml) was stirred atRT for 20 h. Further2-(aminomethyl)-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrochloride chloride, Intermediate 159 (84%, 60 mg, 0.16 mmol) wasadded then the reaction was left to stir at RT for 6 h. The reactionmixture was concentrated in vacuo then the crude material was purifiedby flash column chromatography on C18 (12 g). The column was eluted withMeCN:H₂O+formic acid using the following gradient (% MeCN, columnvolumes): 10%, 1.5 CV; 10-26%, 15 CV; 26-100%, 6 CV; 100%, 1 CV. Thedesired fractions were combined then lyophilised to afford the productas a yellow/green solid (98 mg, 25%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.89 (s, 1H), 9.54 (t, J=5.4 Hz, 1H),8.40-8.36 (m, 1H), 8.22-8.16 (m, 1H), 7.92 (d, J=8.6 Hz, 1H), 7.68 (s,1H), 7.39 (s, 2H), 5.13 (d, J=5.4 Hz, 2H), 4.77-4.62 (m, 4H), 1.46-1.35(m, 6H).

LC/MS (System C): m/z (ESI⁺)=442 [M(³⁵Cl)⁺], 444 [M(³⁷Cl)⁺], R_(t)=1.61min, UV purity=99%.

Example 64—Synthesis of2-[({3-amino-7-bromo-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide

2-(aminomethyl)-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumhydrobromide bromide, Intermediate 83 (388 mg, 0.950 mmol) was added toa mixture of7-bromo-2-(1H-imidazole-1-carbonyl)-5H-pyrrolo[2,3-b]pyrazin-3-amine,Intermediate 164 (243 mg, 0.791 mmol) in DMF (2.5 ml). The resultantmixture was stirred at RT for 19 h. The reaction mixture wasconcentrated in vacuo then the crude material was purified by flashcolumn chromatography on C18 (30 g). The column was eluted withMeCN:H₂O+formic acid using the following gradient (% MeCN, columnvolumes): 5%, 1.5 CV; 5-27%, 12 CV; 27-50%, 3 CV; 50-83%, 1.5 CV;83-100%, 0.5 CV; 100%, 1 CV. The desired fractions were combined thenconcentrated in vacuo to afford the product as a yellow/orange solid(391 mg, 87%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.91 (s, 1H), 9.49 (t, J=5.4 Hz, 1H),8.49-8.42 (m, 1H), 8.20 (dd, J=8.6, 1.2 Hz, 1H), 8.01 (d, J=8.6 Hz, 1H),7.72 (s, 1H), 7.37 (s, 2H), 5.15 (d, J=5.4 Hz, 2H), 4.76 (q, J=7.2 Hz,2H), 4.70 (q, J=7.1 Hz, 2H), 1.45-1.40 (m, 6H).

LC/MS (System C): m/z (ESI⁺)=486 [M(⁷⁹Br)⁺], 488 [M(⁸¹Br)⁺], R_(t)=1.67min, UV purity=100%.

Example 65—Synthesis of2-[({3-amino-7-chloro-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

A mixture of2-[({3-amino-7-chloro-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumchloride, Example 63 (92 mg, 0.19 mmol) and CDI (47 mg, 0.29 mmol) inDMF (2 ml) was stirred at RT for 1.5 h. Additional CDI (40 mg, 0.25mmol) and DMF (1 ml) were added then the reaction was stirred at RT for15 min. The reaction mixture was then added to(2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl](piperidin-4-yl)amino}hexane-1,2,3,4,5-pentoldihydrochloride, Intermediate 112 (145 mg, 0.289 mmol) and rinsed inwith DMF (0.5 ml). The resultant mixture was stirred at RT for 18 h. Thereaction mixture was concentrated in vacuo then the crude material waspurified by flash column chromatography on C18 (30 g). The column waseluted with MeCN:H₂O+TFA using the following gradient (% MeCN, columnvolumes): 5%, 1.5 CV; 5-32%, 28 CV; 52-100%, 2 CV; 100%, 2 CV. Thedesired fractions were combined then lyophilised to afford ayellow/brown solid (29 mg). The material thus obtained was furtherpurified by flash column chromatography on C18 (12 g). The column waseluted with MeCN:H₂O+TFA using the following gradient (% MeCN, columnvolumes): 0%, 1.5 CV; 0-23%, 13 CV; 23-40%, 5 CV. The desired fractionswere combined then lyophilised to afford a yellow/orange solid (16 mg,7.6%).

¹H NMR (500 MHz, CD₃OD) δ 8.18-8.15 (m, 1H), 8.11 (d, J=8.6 Hz, 1H),7.82-7.77 (m, 1H), 7.43 (s, 1H), 5.20 (s, 2H), 4.86-4.81 (m, 4H+HDO),4.37-3.36 (m, 20H), 3.08-2.92 (m, 1H), 2.40-1.66 (m, 4H), 1.64-1.55 (m,6H).

LC/MS (System C): m/z (ESI⁺)=852 [M(³⁵Cl)⁺], 854 [M(³⁷Cl)⁺], R_(t)=0.99min, UV purity=99%.

Example 66—Synthesis of2-[({3-amino-7-bromo-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-iumtrifluoroacetic acid trifluoroacetate

A mixture of2-[({3-amino-7-bromo-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-iumbromide, Example 64 (190 mg, 0.335 mmol) and CDI (81 mg, 0.50 mmol) inDMF (2 ml) was stirred at RT for 2 h. Additional CDI (60 mg, 0.37 mmol)was added then the reaction was left to stir at RT for 0.5 h. Thereaction mixture was added to(2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl](piperidin-4-yl)amino}hexane-1,2,3,4,5-pentoldihydrochloride, Intermediate 112 (90%, 224 mg, 0.402 mmol) and rinsedin with DMF (1 ml). The resultant mixture was left to stir at RT for 18h. The reaction mixture was concentrated in vacuo then the crudematerial was purified in 2 batches by flash column chromatography on C18(30 g). The column was eluted with MeCN:H₂O+TFA using the followinggradient (% MeCN, column volumes): 2%, 1.5 CV; 2-22%, 15 CV; 22-45%, 6CV; 45-100%, 2 CV. The desired fractions from both columns were combinedthen lyophilised to afford the product as a yellow solid (36 mg, 10%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.90 (d, J=2.4 Hz, 1H), 9.51 (t, J=5.3 Hz,1H), 8.31-8.09 (m, 3H), 7.76-7.71 (m, 2H), 7.37 (s, 2H), 5.69-5.43 (m,2H), 5.16 (d, J=5.3 Hz, 2H), 4.93-4.37 (m, 14H), 4.10-3.91 (m, 2H),3.87-3.76 (m, 1H), 3.75-3.56 (m, 6H), 3.53-3.40 (m, 8H), 3.20-3.14 (m,1H), 2.91-2.76 (m, 1H), 2.26-1.55 (m, 4H), 1.47-1.41 (m, 6H).

LC/MS (System C): m/z (ESI⁺)=896 [M(⁷⁹Br)⁺], 898 [M(⁸¹Br)⁺], R_(t)=1.02min, UV purity=100%.

Example 67—Synthesis of2-[({3-amino-7-cyano-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-methoxy-1H-1,3-benzodiazol-3-iumformate

TFA (991 μl, 13.0 mmol) was added to a solution of3-amino-7-cyano-5-{[2-(trimethylsilyl)ethoxy]methyl}-5H-pyrrolo[2,3-b]pyrazine-2-carboxylate,Intermediate 166 (150 mg, 0.43 mmol) in CH₂Cl₂ (2 ml). The resultantmixture was stirred at RT for 4.5 h. The reaction mixture wasconcentrated in vacuo, azeotroped with toluene (2×5 ml), then dried invacuo to afford a red/orange solid (114 mg). A portion (83 mg) of thesolid thus obtained was dissolved in MeOH (3 ml). Aqueous NaOH solution(5.0 M, 0.67 ml, 3.4 mmol) was added then the resulting mixture washeated at 60° C. for 1 h then at 80° C. for 1.5 h. The reaction mixturewas allowed to cool to RT then filtered. The collected solid was washedwith water then dried in vacuo to afford a brown solid (60 mg). Thesolid thus obtained was dissolved in DMF (1 ml) then CDI (78 mg, 0.48mmol) and imidazole hydrochloride (25 mg, 0.24 mmol) were added. Thereaction was stirred at RT for 10 min. Water (3 ml) was added then thereaction was stirred at RT for 5 min. The solid was collected byfiltration, washed with water, then dried in vacuo to afford a brownsolid (39 mg). The solid thus obtained was dissolved in DMF (1 ml) then2-(aminomethyl)-1,3-diethyl-6-methoxy-1H-1,3-benzodiazol-3-ium iodide,Intermediate 36 (45 mg, 0.13 mmol) was added. The resultant mixture wasstirred at RT for 2.5 h then concentrated in vacuo. The crude materialwas purified by flash column chromatography on C18 (12 g). The columnwas eluted with MeCN:H₂O+0.1% TFA using the following gradient (% MeCN,column volumes): 2%, 2 CV; 2-37%, 18 CV; 37-48%, 1 CV; 48-89%, 3 CV;89-100%, 1 CV; 100% 2 CV. The desired fractions were combined andlyophilised. The material thus obtained was further purified bypreparative HPLC (Method A). The desired fractions were combined andlyophilised to afford the product as a yellow solid (3.5 mg, 1.7%).

¹H NMR (500 MHz, DMSO-d₆) δ 9.41 (t, J=5.0 Hz, 1H), 8.34 (s, 1H), 8.23(s, 1H), 7.89 (d, J=9.1 Hz, 1H), 7.51 (d, J=2.3 Hz, 1H), 7.21 (dd,J=9.1, 2.3 Hz, 1H), 7.09 (s, 2H), 5.01 (d, J=5.4 Hz, 2H), 4.66-4.56 (m,4H), 3.84 (s, 3H), 1.38-1.28 (m, 6H).

LC/MS (System C): m/z (ESI⁺)=419 [M⁺], R_(t)=1.85 min, UV purity=99%.

C. BIOLOGICAL EXAMPLES Example 68—Short Circuit Current Assay toDetermine ENaC Blocker Potency in Human Bronchial Epithelial Cells

Cell Culture

Human bronchial epithelial cells (HBECs) (Lonza, UK) were cultured usinga modification of the method described by Coote et al, (2008). Cellswere seeded into plastic T-75 flasks and grown in Bronchial EpithelialCell Growth Medium (BEGM) (Lonza, UK) supplemented with bovine pituitaryextract (52 ng/mL), hydrocortisone (0.5 μg/mL), human recombinantEpidermal Growth Factor (0.5 ng/mL), epinephrine (0.5 ng/mL),transferrin (10 ng/mL), insulin (5 ng/mL), retinoic acid (0.1 ng/mL),triiodothyronine (6.5 ng/mL), gentamycin (50 μg/mL) and amphotericin-B(50 ng/mL). Medium was changed every 48 hours until cells were 90%confluent. Cells were then passaged and seeded (8.25×10⁵ cells/insert)onto polycarbonate Snapwell™ inserts (Costar, UK) in differentiationmedia containing 50% DMEM in BGEM with the same supplements as above butwithout triiodothyronine and a final retinoic acid concentration of 50nM (all-trans retinoic acid; Sigma-Aldrich, UK). Cells were maintainedsubmerged for the first 7 days in culture after which time they wereexposed to an apical air interface for the remainder of the cultureperiod. From the first day of establishment of an ALI, HBEC were fedwith a DMEM:HAMS F-12 (1:1) media containing 2% Ultroser G (PallBioSepra, France) with gentamycin (50 μg/mL) and amphotericin B (50ng/mL). Cells were used for short-circuit current assay between days14-21 after the establishment of the ALI. At all stages of culture,cells were maintained at 37° C. in 5% CO₂ in an air incubator.

Short-Circuit Current (ISC) Measurements

Snapwell inserts were mounted in Costar Vertical Diffusion Chambers(Costar, UK) and were bathed with continuously gassed Ringer solution(5% CO₂ in O₂; pH 7.4) maintained at 37° C. containing (in mM): 120NaCl, 25 NaHCO₃, 3.3 KH₂PO₄, 0.8 K₂HPO₄, 1.2 CaCl₂, 1.2 MgCl₂ and 10glucose. The solution osmolarity was always between 280-300 mOsm/kg H₂Ofor all physiological salt solutions used. Cells were voltage clamped to0 mV (model EVC4000, WPI). Transepithelial resistance (RT) was measuredby applying a 2 mV pulse at 30 s intervals and calculating RT by Ohm'slaw. Data were recorded using a PowerLab workstation (ADInstruments,UK).

ENaC blocker compounds were added to the apical chamber from a 1000-foldstock solution (prepared in DMSO) to achieve a cumulative concentrationresponse in terms of the inhibition of the basal ISC. At the completionof the concentration response, a supra-maximal concentration ofamiloride (10 μM) was added. The concentration of test compound thatinduced a 50% inhibition of the total amiloride-sensitive ISC (IC₅₀) wascalculated using GraphPad Prism v6.05. The results are presented inTable 2, from which it can be seen that the compounds of the presentinvention have ENaC inhibiting activity.

TABLE 2 Averages. ENaC Example No. IC₅₀ (nM) Avg 1 33 2 43 3 19 4 8 5 96 5 7 3 8 9 9 48 10 199 11 22 12 5 13 4 14 11 15 198 16 10 17 15 18 545019 8 20 4 21 2 22 8 23 4 24 6 25 2 26 1 27 14 28 7 29 5 30 6 31 1 32 333 3 34 160 35 5 36 3 37 NR 38 9 39 15 40 7 41 11 42 10 43 51 44 21 4514 46 8 47 5 48 18 49 7 50 35 51 38 52 69 53 202 54 49 55 78 56 15 57 3658 78 59 171 60 22 61 43 62 24 63 133 64 244 65 49 66 199 67 54 NR—notrecorded

Example 69—Bronchoalveolar Lavage (BAL) Procedure

A 0.1 mg/mL solution of ENaC inhibitor in 5% dextrose was administeredintratracheally to a rat weighing 225-250 g. A volume of 1 mL/Kg wasused. After 6 hours, lungs were lavaged with 3×4 mL of sterile saline. A1 mL aliquot was subsequently snap frozen. Lungs were excised, weighedand snap frozen. Compound levels in the BAL and lung tissue weresubsequently determined using LC/MS/MS bioanalysis.

The results are presented in Table 3 and demonstrate that significantamounts of the compounds of the invention persisted in the lungs 6 hoursafter administration.

TABLE 3 BAL@ 6 hours Example No. (ng/mL) 50 260 52 302 54 139 55 284 56283 57 283 58 256 59 197 60 176 61 167 62 140

Example 70—Sheep Mucociliary Clearance (MCC)

MCC was measured in conscious sheep as previously described (Coote etal., 2009; Hirsh et al., 2008). Briefly, adult ewes (25-45 kg) wererestrained in an upright position in specialized body harness inmodified carts. The head of the animal was immobilized, and after localanesthesia of the nasal passage was induced with 2% lidocaine, theanimals were nasally intubated with a standard endotracheal tube (7.5 mmdiameter, Mallinckrodt, St. Louis, Mo.). Test compounds and vehicle weredelivered as nebulized aqueous solution via the endotracheal tube as avolume of 3 mL. All aerosols were generated using a Raindrop Nebulizer(Nellcor Puritan Bennett, Carlsbad, Calif.) which produces a dropletwith a mass median aerodynamic diameter (MMAD) of approximately 1.1 μm.The output of the nebulizer was connected to a T-piece, with one endattached to a respirator (Harvard Apparatus Inc., Holliston, Mass.). Thesystem was activated for 1 second at the onset of the inspiratory cycleof the respirator, which was set at an inspiratory/expiratory ratio of1:1 and a rate of 20 breaths min-1. Aerosolized technetium labeledsulfur colloid (99mTc-SC) was used to measure the effects of the variousdoses of test compounds or control on MCC. Approximately 20 millicurieof 99mTc-SC in a total volume of 2 mL of sterile saline was placed inthe nebulizer. A tidal volume of 500 mL was used to deliver the 99mTc-SCfor 3 minutes. A gamma camera (Dyna Cam, Picker Corp., Nothford, Conn.)integrated with a computer was used to record and analyze the clearanceof 99mTc-SC over 2 hours. After 99mTc-SC nebulization, the animals wereimmediately extubated and positioned in their natural upright positionunderneath the gamma camera so that the field of image was perpendicularto the animals' spinal cord. After acquisition of a baseline image,serial images were obtained over a 2 hour period at 5 minute intervalsfor the first hour and then every 15 minutes for the next hour. Allimages were obtained and stored in the computer for analysis. An ‘areaof interest’ was traced over the image corresponding to the right lungof the animals, and counts were recorded. The left lung was excludedfrom analysis because its corresponding image is superimposed over thestomach and counts could be affected by swallowed radiolabeled mucus.The counts were corrected for decay and expressed as a percentage ofradioactivity cleared relative to the baseline image (% cleared).Differences in clearance of 99mTc-SC were compared at both 60 and 120min after radioaerosol administration.

The results are presented in FIGS. 1-13 , all of which show that, forall of the compounds tested, the amount of 99mTc-SC cleared over a 120minute period, 4 hours after administration of test compound wassignificantly increased compared with the amount cleared when the sheepwas treated with water. Repeat dosing was carried out for the compoundof Example 50. In this case, the compound was administered at a dose of3 μg/kg twice daily (BiD) at 12 hour intervals for a total of 7 doses(i.e. over 3.5 days). Measurement of the amount of 99mTc-SC cleared overa 120 minute period was begun 4 hours after administration of the finaldose of the test compound. The results are presented in FIG. 3B, whichcompares a single 13 μg/kg dose, 3 μg/kg BiD dosing and a single dose ofwater.

The sheep mucociliary clearance model described above is a model forstudying the clearance of mucus and is therefore a model for theeffectiveness of test compounds in diseases and conditions characterisedby a build-up of mucus in the lungs, for example cystic fibrosis,chronic bronchitis, bronchiectasis, severe asthma and primary ciliarydyskinesia. Therefore, the results presented in FIGS. 1-13 indicate thatthe compounds tested are likely to be of use in the treatment ofconditions of this type, as well as other diseases and conditionsmediated by ENaC.

The inventors have also compared in this model certain compounds of thepresent invention with compounds in which the pyrrolopyrazine moiety isreplaced with the conventional 6-chloro-3,5-diaminopyrazine moiety whichoccurs in the majority of prior art compounds as discussed above, butwhich are otherwise structurally identical. They were able to show thatin the sheep MCC model the tested compounds of general formula (I)showed a significant increase in mucociliary clearance compared with thecorresponding 6-chloro-3,5-diaminopyrazine compounds. This indicatesthat compounds of the present invention have superior activity in vivocompared with prior art compounds.

REFERENCES

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The invention claimed is:
 1. A method for the treatment of a disease orcondition in a patient, wherein the method comprises administering tothe patient in need thereof an effective amount of a compound of formula(I):

or a pharmaceutically acceptable salt, stereoisomer, or tautomerthereof, wherein: R¹ is H, halo, R¹², C(═NR⁹)NR¹²R¹³, C(O)NR¹²R¹³,C(O)OR¹², OR¹², S(O)₂R¹², Q¹R¹², Q¹C(═NR⁷)NR¹²R¹³, Q¹C(O)NR¹²R¹³,Q¹C(O)OR¹², Q¹OR¹², Q¹S(O)₂R¹², Q¹Q²C(═NR⁹)NR¹²R¹³, Q¹Q²C(O)NR¹²R¹³,Q¹Q²C(O)OR¹², Q¹Q²OR¹², or L¹R¹⁰; R² is C₁₋₁₀ alkyl, wherein the C₁₋₁₀alkyl is optionally substituted with one or more substituentsindependently selected from the group consisting of halo, C(O)N(R⁷)R⁸,C(O)OR⁷, N(R⁷)R⁸, OR⁷, SH, cycloalkyl, heterocyclyl, aryl, andheteroaryl, and further wherein one or more —CH₂— groups are optionallyand independently replaced by —N(R⁷)—, —O—, or —S—, provided thatadjacent —CH₂— groups are not so replaced; R³ is C₁₋₁₀ alkyl, whereinthe C₁₋₁₀ alkyl is optionally substituted with one or more substituentsindependently selected from the group consisting of halo, C(O)N(R⁷)R⁸,C(O)OR⁷, N(R⁷)R⁸, OR⁷, SH, cycloalkyl, heterocyclyl, aryl, andheteroaryl, and further wherein one or more —CH₂— groups are optionallyand independently replaced by —N(R⁷)—, —O—, or —S—, provided thatadjacent —CH₂— groups are not so replaced; R⁴ is H, halo, cyano, C₁₋₆alkyl, C(O)N(R¹⁶)R¹⁷, or C(O)OR¹⁶, wherein the C₁₋₆ alkyl is optionallysubstituted with one or more substituents independently selected fromthe group consisting of halo, N(R⁷)R⁸, and OR⁷; R⁵ is H or methyl; L¹ is—Z¹—, -Q¹-, —Z¹Q¹-, -Q¹Z¹—, —Z¹Q¹Z²—, -Q¹Q²-, -Q¹Q²Z¹—, -Q¹Q²Z¹Q³Z²—,—Z¹Q¹OQ²OQ³-, —OZ¹—, -OQ¹-, —OZ¹Q¹-, —OQ¹Z¹—, —OZ¹Q¹Z²—, —OQ¹Q²-,—OQ¹Q²Z¹—, —OQ¹Q²Z¹Q³Z²—, —OZ¹Q¹OQ²OQ³-, —Z¹N(R⁷)Z²—, -Q¹Z¹N(R⁷)Z²—,—Z¹N(R⁷)Z²Q¹-, -Q¹Z¹N(R⁷)Z²Q²Z³—, —Z¹O(CH₂CH₂O)_(n)Z²—, —Z¹O(CH₂CH₂O)Q¹-, —Z¹O(CH₂CH₂O)_(n)Z²Q¹-, —Z¹O(CH₂CH₂O)Q¹Z²—, -Q¹Z¹O(CH₂CH₂O)_(n)Z²—,-Q¹Z¹O(CH₂CH₂O)Q¹-, -Q¹Z¹O(CH₂CH₂O)_(n)Z²Q¹-, —Z¹O(CH₂CH₂O)_(n)Z²Q¹Z³—,—C(O)Z¹—, —C(O)Q¹-, —C(O)Z¹Q¹-, —C(O)Z¹Q¹Z²—, —C(O)Q¹Z¹—, —C(O)Q¹Q²-,—C(O)Q¹Q²Z¹—, —C(O)Q¹N(R⁷)C(O)Z¹—, —C(O)Q¹N(R⁷)C(O)Z¹Q²-,—C(O)Q¹N(R⁷)C(O)Z¹Q²Q³-, —C(O)Q¹N(R⁷)C(O)Z¹Q²Z²—, —C(O)Z¹Q¹OQ²OQ³-,—C(O)N(R⁷)Z¹—, —C(O)N(R⁷)Q¹-, —C(O)N(R⁷)Z¹Q¹-, —C(O)N(R⁷)Z¹Q¹Z²—,—C(O)N(R⁷)Q¹Z¹—, —C(O)N(R⁷)Q¹Q²-, —C(O)N(R⁷)Q¹Q²Z¹—,—C(O)N(R⁷)Z¹Q¹Q²Z²—, —C(O)N(R⁷)Z¹O(CH₂CH₂O)_(n)Z²—, —C(O)N(R⁷)Z¹O(CH₂O)Z²—, —C(O)N(R⁷)Z¹Q¹Z²N(R⁸)Z³—, —C(O)N(R⁷)Z¹N(R⁸)Z²—,—C(O)N(R⁷)Q¹Z¹N(R⁸)Z²—, —C(O)N(R⁷)Z¹Q¹OQ²OQ³-, —C(O)N(R⁷)Z¹Q¹OQ²OQ³Z²—,—Z¹C(O)N(R⁷)Z²—, —Z¹C(O)N(R⁷)Q¹-, —Z¹C(O)N(R⁷)Z²Q¹-, —Z¹C(O)N(R⁷)Q¹Z²—,—Z¹C(O)N(R⁷)Q¹Q²-, —Z¹C(O)Q¹-, —Z¹C(O)Q¹Z²—, —Z¹C(O)Q¹Q²-,—Z¹C(O)N(R⁷)Q¹Q²Z²—, —C(O)OZ¹—, —C(O)OQ¹-, —C(O)OZ¹Q¹-, —C(O)OZ¹Q¹Z²—,—C(O)OQ¹Z¹—, —C(O)OQ¹Q²-, —C(O)OQ¹Q²Z¹—, -Q¹C(O)Q²-, -Q¹C(O)Z¹—,-Q¹C(O)Q²Z¹—, -Q¹C(O)Q²Q³-, -Q¹C(O)Z¹Q²-, -Q¹C(O)Q²Q³Z¹—,—C(═NR⁹)N(R⁷)Z¹—, —C(═NR⁹)N(R⁷)Q¹-, —C(═NR⁹)N(R⁷)Z¹Q¹-,—C(═NR⁹)N(R⁷)Z¹Q¹Z², —C(═NR⁹)N(R⁷)Q¹Z¹—, —C(═NR⁹)N(R⁷)Q¹Q²-, or—C(═NR⁹)N(R⁷)Q¹Q²Z¹—; R¹⁰ is H, C(O)OR⁷, N(R⁷)R⁸, N(R⁷)C(═NR⁹)N(R⁸)₂,N(R⁷)C(O)OR⁸, or OR⁷; or R¹⁰ is N(R⁷)C(O)—C₁₋₃ alkylene-N(R⁸)₃ ⁺; R¹² isH, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₈ cycloalkyl, or 3- to8-membered heterocyclyl, wherein each C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆alkynyl is optionally and independently substituted with one or moresubstituents independently selected from the group consisting of halo,C(O)N(R⁷)R⁸, C(O)OR⁷, N(R⁷)R⁸, and OR⁷, and further wherein each C₃₋₈cycloalkyl and 3- to 8-membered heterocyclyl is optionally andindependently substituted with one or more substituents independentlyselected from the group consisting of oxo, halo, C(O)N(R⁷)R⁸, C(O)OR⁷,N(R⁷)R⁸, and OR⁷; R¹³ is H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₈cycloalkyl, or 3- to 8-membered heterocyclyl, wherein each C₁₋₆ alkyl,C₂₋₆ alkenyl, and C₂₋₆ alkynyl is optionally and independentlysubstituted with one or more substituents independently selected fromthe group consisting of halo, C(O)N(R⁷)R⁸, C(O)OR⁷, N(R⁷)R⁸, and OR⁷,and further wherein each C₃₋₈ cycloalkyl and 3- to 8-memberedheterocyclyl is optionally and independently substituted with one ormore substituents independently selected from the group consisting ofoxo, halo, C(O)N(R⁷)R⁸, C(O)OR⁷, N(R⁷)R⁸, and OR⁷; R⁷ is H or C₁₋₁₂alkyl, wherein the C₁₋₁₂ alkyl is optionally substituted with one ormore substituents independently selected from the group consisting ofhalo and OH; R⁸ is H or C₁₋₁₂ alkyl, wherein the C₁₋₁₂ alkyl isoptionally substituted with one or more substituents independentlyselected from the group consisting of halo and OH; or R⁷ and R⁸,together with any nitrogen atom to which they are attached, form a 5- or6-membered heterocyclic ring optionally containing one or moreadditional heteroatoms selected from the group consisting of nitrogen,oxygen, and sulfur; or two R⁸, together with any nitrogen atom to whichthey are attached, form a 5- or 6-membered heterocyclic ring optionallycontaining one or more additional heteroatoms selected from the groupconsisting of nitrogen, oxygen, and sulfur; R⁹ is H or C₁₋₆ alkyl; Q¹ iscarbocyclyl, heterocyclyl, aryl, or heteroaryl, wherein each aryl andheteroaryl is optionally and independently substituted with one or moresubstituents independently selected from the group consisting of halo,C₁₋₄ alkyl, C₁₋₄ haloalkyl, C(O)NR¹⁵R¹⁶, C(O)OR¹⁵, NR¹⁵R¹⁶, and OH, andfurther wherein each carbocyclyl and heterocyclyl is optionally andindependently substituted with one or more substituents independentlyselected from the group consisting of oxo, halo, C₁₋₄ alkyl, C₁₋₄haloalkyl, C(O)NR¹⁵R¹⁶, C(O)OR's, NR¹⁵R¹⁶, and OH; Q² is carbocyclyl,heterocyclyl, aryl, or heteroaryl, wherein each aryl and heteroaryl isoptionally and independently substituted with one or more substituentsindependently selected from the group consisting of halo, C₁₋₄ alkyl,C₁₋₄ haloalkyl, C(O)NR¹⁵R¹⁶, C(O)OR¹⁵, NR¹⁵R¹⁶, and OH, and furtherwherein each carbocyclyl and heterocyclyl is optionally andindependently substituted with one or more substituents independentlyselected from the group consisting of oxo, halo, C₁₋₄ alkyl, C₁₋₄haloalkyl, C(O)NR¹⁵R¹⁶, C(O)OR¹⁵, NR¹⁵R¹⁶, and OH; Q³ is carbocyclyl,heterocyclyl, aryl, or heteroaryl, wherein each aryl and heteroaryl isoptionally and independently substituted with one or more substituentsindependently selected from the group consisting of halo, C₁₋₄ alkyl,C₁₋₄ haloalkyl, C(O)NR¹⁵R¹⁶, C(O)OR¹⁵, NR¹⁵R¹⁶, and OH, and furtherwherein each carbocyclyl and heterocyclyl is optionally andindependently substituted with one or more substituents independentlyselected from the group consisting of oxo, halo, C₁₋₄ alkyl, C₁₋₄haloalkyl, C(O)NR¹⁵R¹⁶, C(O)OR's, NR¹⁵R¹⁶, and OH; Z¹ is C₁₋₁₂ alkylene,C₂₋₁₂ alkenylene, or C₂₋₁₂ alkynylene, wherein the C₁₋₁₂ alkylene, C₂₋₁₂alkenylene, and C₂₋₁₂ alkynylene is optionally substituted by one ormore substituents independently selected from the group consisting ofhalo, C(O)NR¹⁵R¹⁶, C(O)OR¹⁵, NR¹⁵R¹⁶, and OH; Z² is C₁₋₁₂ alkylene,C₂₋₁₂ alkenylene, or C₂₋₁₂ alkynylene, wherein the C₁₋₁₂ alkylene, C₂₋₁₂alkenylene, and C₂₋₁₂ alkynylene is optionally substituted by one ormore substituents independently selected from the group consisting ofhalo, C(O)NR¹⁵R¹⁶, C(O)OR¹⁵, NR¹⁵R¹⁶, and OH; Z³ is C₁₋₁₂ alkylene,C₂₋₁₂ alkenylene, or C₂₋₁₂ alkynylene, wherein the C₁₋₁₂ alkylene, C₂₋₁₂alkenylene, and C₂₋₁₂ alkynylene is optionally substituted by one ormore substituents independently selected from the group consisting ofhalo, C(O)NR¹⁵R¹⁶, C(O)OR¹⁵, NR¹⁵R¹⁶, and OH; R¹⁵ is H or C₁₋₆ alkyl;R¹⁶ is H or C₁₋₆ alkyl; R¹⁷ is H or C₁₋₆ alkyl; or R¹⁵ and R¹⁶, togetherwith the nitrogen atom to which they are attached, form a 5- or6-membered heterocyclic ring, wherein the 5- or 6-membered heterocyclicring optionally contains one or more additional heteroatomsindependently selected from the group consisting of nitrogen, oxygen,and sulfur; or R¹⁶ and R¹⁷, together with the nitrogen atom to whichthey are attached, form a 5- or 6-membered heterocyclic ring, whereinthe 5- or 6-membered heterocyclic ring optionally contains one or moreadditional heteroatoms independently selected from the group consistingof nitrogen, oxygen, and sulfur; n is 1, 2, 3, 4, 5, or 6; and X⁻ is ananion; with the proviso that if R¹⁰ is N(R⁷)C(O)—C₁₋₃ alkylene-N(R⁸)₃ ⁺or N(R⁸)₃ ⁺, then the compound of formula (I) has an additional X⁻;wherein the disease or condition is mediated by epithelial sodiumchannel (ENaC) activity; and wherein the disease or condition isselected from the group consisting of an ocular condition, a respiratorydisease, a respiratory condition, and a skin condition.
 2. The methodaccording to claim 1, wherein the compound is of formula (IA) or formula(IB):

or a pharmaceutically acceptable salt, stereoisomer, or tautomerthereof.
 3. The method according to claim 1, wherein R¹ is H, halo, R¹²,C(O)OR¹², or OR¹².
 4. The method according to claim 1, wherein R¹ isL¹R¹⁰.
 5. The method according to claim 4, wherein L¹ is —Z¹—, -Q¹-,-Q¹Z¹—, -Q¹Q²-, -Q¹Q²Z¹—, —OZ¹—, —C(O)Q¹-, —C(O)Q¹Z¹—, —C(O)N(R⁷)Z¹—,—C(O)N(R⁷)Q¹-, —C(O)N(R⁷)Z¹Q¹-, —C(O)N(R⁷)Q¹Z¹—, —C(O)N(R⁷)Z¹Q¹Q²Z²—,—C(O)N(R⁷)Z¹O(CH₂CH₂O)_(n)Z²—, or —C(O)N(R⁷)Z¹Q¹Z²N(R⁸)Z³.
 6. The methodaccording to claim 4, wherein: R¹⁰ is H, C(O)OR⁷, N(R⁷)R⁸,N(R⁷)C(═NR⁹)N(R⁸)₂, N(R⁷)C(O)OR⁸, or OR⁷; or R¹⁰ is N(R⁷)C(O)—C₁₋₃alkylene-N(R⁸)₃ ⁺ or N(R⁸)₃ ⁺.
 7. The method according to claim 6,wherein: a) L¹ is -Q¹- or —C(O)N(R⁷)Q¹-; Q¹ is carbocyclyl; and R¹⁰ isC(O)OR⁷; or b) L¹ is -Q¹- or —C(O)N(R⁷)Q¹-; Q¹ is heterocyclyl, whereinthe heterocyclyl is linked to R¹⁰ via a ring nitrogen atom; and R¹⁰ isC(O)OR⁷; or c) L¹ is -Q¹Q²-; Q² is carbocyclyl; and R¹⁰ is C(O)OR⁷; ord) L¹ is -Q¹Q²-; Q² is heterocyclyl, wherein the heterocyclyl is linkedto R¹⁰ via a ring carbon atom; and R¹⁰ is C(O)OR⁷; or e) L¹ is —Z¹—,—OZ¹—, —C(O)Q¹Z¹—, —C(O)N(R⁷)Z¹—, —C(O)N(R⁷)Z¹Q¹Q²Z²—,—C(O)N(R⁷)Z¹O(CH₂CH₂O)_(n)Z²—, or —C(O)N(R⁷)Z¹Q¹Z²N(R⁸)Z³—; and R¹⁰ isN(R⁷)R⁸, N(R⁷)C(═NR⁹)N(R⁸)₂, or N(R⁷)C(O)OR⁸; or f) L¹ is —C(O)Q¹-,—C(O)N(R⁷)Q¹-, or —C(O)N(R⁷)Z¹Q¹-; Q¹ is carbocyclyl; and R¹⁰ isN(R⁷)R⁸, N(R⁷)C(═NR⁹)N(R⁸)₂, or N(R⁷)C(O)OR⁸; or g) L¹ is —C(O)Q¹-,—C(O)N(R⁷)Q¹-, or —C(O)N(R⁷)Z¹Q¹-; Q¹ is heterocyclyl, wherein theheterocyclyl is linked to R¹⁰ via a ring carbon atom; and R¹⁰ isN(R⁷)R⁸, N(R⁷)C(═NR⁹)N(R⁸)₂, or N(R⁷)C(O)OR⁸.
 8. The method according toclaim 7, wherein: a) R¹⁰ is N(R⁷)R⁸; and R⁷ is CH₂[CH(OH)]₄—CH₂OH; or b)R¹⁰ is N(R⁷)R⁸; and R⁸ is CH₂[CH(OH)]₄—CH₂OH; or c) R¹⁰ is N(R⁷)R⁸; R⁷is CH₂[CH(OH)]₄—CH₂OH; and R⁸ is CH₂[CH(OH)]₄—CH₂OH.
 9. The methodaccording to claim 7, wherein: R¹⁰ is N(R⁷)C(═NR⁹)N(R⁸)₂; R⁷ is H orC₁₋₄ alkyl; at least one R⁸ is CH₂[CH(OH)]₄—CH₂OH; and R⁹ is H or C₁₋₆alkyl.
 10. The method according to claim 7, wherein R¹⁰ isN(CH₂[CH(OH)]₄—CH₂OH)₂.
 11. The method according to claim 4, wherein: a)L¹ is —OZ¹—; Z¹ is C₁₋₄ alkylene; and R¹⁰ is H; or b) L¹ is -Q¹- or—C(O)N(R⁷)Q¹-; Q¹ is a nitrogen-containing heterocyclyl, wherein theheterocyclyl is linked to R¹⁰ via a ring nitrogen atom; and R¹⁰ is H; orc) L¹ is -Q¹Q²-; Q² is a nitrogen-containing heterocyclyl, wherein theheterocyclyl is linked to R¹⁰ via a ring nitrogen atom; and R¹⁰ is H; ord) L¹ is —Z¹—, -Q¹-, -Q¹Z¹—, -Q¹Q²-, -Q¹Q²Z¹—, —OZ¹—, —OQ¹Z¹—,—OQ¹Q²Z¹—, —C(O)Z¹—, —C(O)Q¹Z¹—, —C(O)Q¹Q²Z¹—, —C(O)N(R⁷)Z¹—,—C(O)N(R⁷)Q¹Z¹—, —C(O)N(R⁷)Q¹Q²Z¹—, —C(O)OZ¹—, —C(O)OQ¹Z¹—, —C(O)OQ¹Q²-,—C(O)OQ¹Q²Z¹—, —C(═NR⁹)N(R⁷)Z¹—, —C(═NR⁹)N(R⁷)Q¹Z¹—, or—C(═NR⁹)N(R⁷)Q¹Q²Z¹—; and R¹⁰ is H; or e) L¹ is -Q¹Z¹—, —Z¹Q¹Z²—,-Q¹Q²Z¹—, —OQ¹Z¹—, —OZ¹Q¹Z²—, —OQ¹Q²Z¹—, —Z¹O(CH₂CH₂O)_(n)Q¹Z²—,-Q¹Z¹O(CH₂CH₂O) Z²—, —Z¹O(CH₂CH₂O)_(n)Z²Q¹Z³—, —C(O)Z¹Q¹Z²—, —C(O)Q¹Z¹—,—C(O)Q¹Q²Z¹—, —C(O)Q¹N(R⁷)C(O)Z¹Q²Z²—, —C(O)N(R⁷)Z¹Q¹Z²—,—C(O)N(R⁷)Q¹Z¹—, —C(O)N(R⁷)Q¹Q²Z¹—, —C(O)N(R⁷)Z¹Q¹Q²Z²—,—C(O)N(R⁷)Z¹Q¹OQ²OQ³Z²—, —Z¹C(O)N(R⁷)Q¹Z²—, —Z¹C(O)Q¹Z²—,—Z¹C(O)N(R⁷)Q¹Q²Z²—, —C(O)OZ¹Q¹Z²—, —C(O)OQ¹Z¹—, —C(O)OQ¹Q²Z¹—,-Q¹C(O)Q²Z¹—, -Q¹C(O)Q²Q³Z¹—, C(═NR⁹)N(R⁷)Z¹Q¹Z², —C(═NR⁹)N(R⁷)Q¹Z¹—, or—C(═NR⁹)N(R⁷)Q¹Q²Z¹—; Q¹ is a nitrogen-containing heterocyclyl, whereinthe heterocyclyl is linked to each of Z¹, Z², and/or Z³ via a ringnitrogen atom; Q² is a nitrogen-containing heterocyclyl, wherein theheterocyclyl is linked to each of Z¹, Z², and/or Z³ via a ring nitrogenatom; Q³ is a nitrogen-containing heterocyclyl, wherein the heterocyclylis linked to each of Z¹, Z², and/or Z³ via a ring nitrogen atom; and R¹⁰is H; or f) L¹ is —CH₂[CH(OH)]₄—CH(OH)—; and R¹⁰ is H.
 12. The methodaccording to claim 1, wherein: R² is unsubstituted C₁₋₄ alkyl; and R³ isunsubstituted C₁₋₄ alkyl.
 13. The method according to claim 1, wherein:a) R⁴ is H; or b) R⁵ is H; or c) R⁴ is H; and R⁵ is H.
 14. The methodaccording to claim 1, wherein X⁻ is chloride, bromide, iodide, sulfate,nitrate, phosphate, formate, acetate, trifluoroacetate, fumarate,citrate, tartrate, oxalate, succinate, mandelate, methanesulfonate, orp-toluenesulfonate.
 15. The method according to claim 1, wherein thecompound has a cation selected from the group consisting of:2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-3-methyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-6-fluoro-3-methyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-chloro-1-ethyl-3-methyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-6-methoxy-3-methyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-3-methyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-3-methyl-6-(trifluoromethoxy)-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-methoxy-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-3-benzyl-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-6-methoxy-3-(2-methoxy-2-oxoethyl)-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-3-(carboxylatomethyl)-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-ium2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-3-(carbamoylmethyl)-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-6-methoxy-3-[2-(methylsulfanyl)ethyl]-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-3-(2-hydroxyethyl)-6-methoxy-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-3-{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}-6-methoxy-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-benzyl-3-methyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-3-benzyl-6-chloro-1-ethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-3-benzyl-1-ethyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-3-benzyl-1-[2-oxo-2-(piperidin-1-yl)ethyl]-1H-1,3-benzodiazol-3-ium;2-[({3-amino-6-methyl-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1-ethyl-6-methoxy-3-methyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-7-methyl-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-chloro-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-7-chloro-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-chloro-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-{[(tert-butoxy)carbonyl]amino}propyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(1-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1H-pyrazol-4-yl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(2-aminoethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-aminopropoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-aminopropyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-(piperidin-4-yl)-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(2-carbamimidamidoethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(3-carbamimidamidopropyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3-{[(tert-butoxy)carbonyl]amino}propyl)carbamoyl]-1,3-diethyl-1H-1,3-benzodiazol-3-iumformic acid; 2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-({1-[(tert-butoxy)carbonyl]piperidin-4-yl}carbamoyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{[(tert-butoxy)carbonyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{[2-(4-{[(tert-butoxy)carbonyl]amino}piperidin-1-yl)ethyl]carbamoyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3-aminopropyl)carbamoyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-[(piperidin-4-yl)carbamoyl]-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-aminopiperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{[2-(4-aminopiperidin-1-yl)ethyl]carbamoyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-5-(2-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}ethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-5-(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-5-(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-{1-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]piperidin-4-yl}-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-(1-{1-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]piperidin-4-yl}-1H-pyrazol-4-yl)-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)carbamoyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-({1-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]piperidin-4-yl}carbamoyl)-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{[2-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidin-1-yl)ethyl]carbamoyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-5-[4-({bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}methyl)piperidine-1-carbonyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3R)-3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}pyrrolidine-1-carbonyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3S)-3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}pyrrolidine-1-carbonyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-{[(1r,4r)-4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}cyclohexyl]carbamoyl}-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-{[(1s,4s)-4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}cyclohexyl]carbamoyl}-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)(methyl)carbamoyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-[(2-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}ethyl)carbamoyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-5-{[(14S,15R,16R,17R)-14,15,16,17,18-pentahydroxy-12-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]-3,6,9-trioxa-12-azaoctadecan-1-yl]carbamoyl}-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-({2-[4′-(2-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}ethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamoyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{[(3S)-3-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)amino]-3-carbamoylpropyl]carbamoyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-{[4-(4-{3-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)amino]-3-carbamoylpropyl}phenyl)butyl]carbamoyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-7-chloro-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-7-bromo-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-7-chloro-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;2-[({3-amino-7-bromo-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium;and2-[({3-amino-7-cyano-5H-pyrrolo[2,3-b]pyrazin-2-yl}formamido)methyl]-1,3-diethyl-6-methoxy-1H-1,3-benzodiazol-3-ium.16. The method according to claim 15, wherein X⁻ is chloride, bromide,iodide, sulfate, nitrate, phosphate, formate, acetate, trifluoroacetate,fumarate, citrate, tartrate, oxalate, succinate, mandelate,methanesulfonate, or p-toluenesulfonate.
 17. The method according toclaim 15, wherein the compound has the cation:


18. The method according to claim 17, wherein X⁻ is chloride, bromide,iodide, sulfate, nitrate, phosphate, formate, acetate, trifluoroacetate,fumarate, citrate, tartrate, oxalate, succinate, mandelate,methanesulfonate, or p-toluenesulfonate.
 19. The method according toclaim 15, wherein the compound has the cation:


20. The method according to claim 19, wherein X⁻ is chloride, bromide,iodide, sulfate, nitrate, phosphate, formate, acetate, trifluoroacetate,fumarate, citrate, tartrate, oxalate, succinate, mandelate,methanesulfonate, or p-toluenesulfonate.
 21. The method according toclaim 1, wherein: (i) the ocular condition is dry eye disease; (ii) therespiratory disease or respiratory condition is selected from the groupconsisting of asthma, bronchiectasis, chronic bronchitis, chronicobstructive pulmonary disease, cystic fibrosis, emphysema, and primaryciliary dyskinesia; and (iii) the skin condition is selected from thegroup consisting of atopic dermatitis, ichthyosis, and psoriasis. 22.The method according to claim 1, wherein the method further comprisesadministering to the patient in need thereof an effective amount of anadditional active agent selected from the group consisting of a β2adrenoreceptor agonist, an antihistamine, dornase alfa, acorticosteroid, a leukotriene antagonist, a cystic fibrosistransmembrane conductance regulator repairing agent, a transmembranemember 16A modulator, and an antibiotic.
 23. A method for the treatmentof a disease or condition in a patient, wherein the method comprisesadministering to the patient in need thereof an effective amount of acompound having the cation:

wherein the disease or condition is mediated by epithelial sodiumchannel (ENaC) activity; and wherein the disease or condition is arespiratory disease or respiratory condition selected from the groupconsisting of asthma, bronchiectasis, chronic bronchitis, chronicobstructive pulmonary disease, cystic fibrosis, emphysema, and primaryciliary dyskinesia.
 24. The method according to claim 23, wherein therespiratory disease or respiratory condition is cystic fibrosis.
 25. Amethod for the treatment of a disease or condition in a patient, whereinthe method comprises administering to the patient in need thereof aneffective amount of a compound having the cation:

wherein the disease or condition is mediated by epithelial sodiumchannel (ENaC) activity; and wherein the disease or condition is arespiratory disease or respiratory condition selected from the groupconsisting of asthma, bronchiectasis, chronic bronchitis, chronicobstructive pulmonary disease, cystic fibrosis, emphysema, and primaryciliary dyskinesia.
 26. The method according to claim 25, wherein therespiratory disease or respiratory condition is cystic fibrosis.